WO2020117002A1 - Method for predicting prognosis of esophageal squamous cell carcinoma - Google Patents

Abstract

The present invention relates to a method for predicting the prognosis of esophageal squamous cell carcinoma.

Description

How to predict the prognosis of esophageal squamous cell carcinoma

The present invention relates to a method for predicting the prognosis of esophageal squamous cell carcinoma.

In the past 20 years, the role of non-coding RNAs (ncRNAs) has been extensively studied. Recently discovered by the main author of the present invention, nc886 is one of the regulatory ncRNAs that affect gene expression patterns. nc886 binds to the target protein and modulates its activity. Target proteins of nc886 known to date are Protein Kinase R (PKR) and Dicer. Another feature of nc886 is that it is transcribed by RNA polymerase III, which is very different from most regulatory ncRNA transcribed by RNA polymerase II. nc886 began to receive attention after the dysregulation was found in various malignant tumors. nc886 is expressed in most normal tissues, but its expression is epigenetically silent in many malignancies such as gastric cancer and acute myeloid leukemia. A prominent phenotypic result that appears when expression of nc886 changes is cell proliferation. Therefore, it was assumed that nc886 was a tumor suppressor, and the silence of nc886 was expected to promote cell proliferation. Nevertheless, when nc886 was treated with anti-sense oligonucleotides in some nc886 ⁺ cell lines derived from the esophagus, stomach, etc., the expression of nc886 was rapidly reduced and cell proliferation was reduced. The above results can be well explained as follows as nc886 is a molecular function that inhibits PKR. PKR is a procytic protein (a protein that induces apoptosis) that is typically activated by viral infection. The rapid decrease in nc886 is sufficient to activate PKR without viral infection. When PKR is activated, it phosphorylates elF2α, thereby stopping the translation of the entire protein and inducing cell death. The significance of nc886 silencing and PKR activation (and thereby apoptosis) in vivo is thought to act as a boundary for removing pre-cancerous cells, such as PKR activation removing virus-infected cells.

Nonetheless, there are cells that do not express nc886 that are clinically detectable or isolated in vitro, and these are cells that bypass the nc886/PKR mediated cell death pathway. Based on the fact that the prognosis of cancer patients with reduced expression of nc886 is poor, it was assumed that nc886 silenced cancer cells proliferate more. As an experimental result supporting this, there is an observation that temporary expression of nc886 in esophageal squamous cell carcinoma (ESCC) and gastric cancer (GC) cells inhibits cell proliferation. All these data hypothesized that nc886 silencing confers growth benefits on cells at certain stages of cancer development. In a situation where nc886 is silenced, it is thought that the growth promoting effect in addition to the PKR-eIF2α cell death pathway also occurs, and these two are separate pathways. When experimentally inducing the silence of nc886, cell death according to the PKR pathway occurs first, so it is not easy to observe the cell growth promoting phenotype due to the silence of nc886.

The dysregulation of cell cycle progression/stoppage in the process of tumor formation is due to the malfunction of several signaling pathways, such as the AKT pathway, which is involved in various cellular functions including cell proliferation, survival and metabolism. In cancer, AKT promotes G1-S metastasis through several mechanisms. The main mechanism is to directly phosphorylate the cyclin-dependent kinase (CDK) inhibitory protein to make it inactive, but another possible mechanism is to regulate the transcription of the CDK inhibitory protein through E2F, or the stability of the CDK inhibitory protein through the ubiquitin-proteasome system. And indirectly. E2F activity is inhibited by AKT, and some CDK inhibitors (such as CDKN2A and CDKN2C) have been reported to be transcriptionally activated by E2F. Therefore, AKT inhibits E2F, thereby weakening the transcription of the CDK inhibitor gene. In addition, crosstalk between AKT and SKP2 has been reported. SKP2, a regulatory component of the SKP1:CUL1:F-box protein complex (SCF), is overexpressed in malignant tumors and instructs to break down CDK inhibitory proteins through SCF E3 ubiquitinase. do.

Esophageal cancer is a generic term for cancer that occurs in the esophagus, and is not one of the most common cancers in Korea. Therefore, measuring a patient's prognosis prediction is one of the most important studies. For example, Korean Patent No. 10-1657033 discloses a method of predicting the survival rate and prognosis of esophageal cancer patients by measuring the protein expression level of V-ATPase subunit V1E1, and Korean Patent Publication No. 10-2008-0007659 The disclosure discloses compositions and methods for the diagnosis of esophageal cancer and esophageal cancer metastasis by detecting specific genes. Esophageal cancer is largely divided into esophageal squamous cell carcinoma and esophageal adenocarcinoma. Among them, esophageal squamous cell carcinoma is a cancer originating from flat cells, which are the main cells that make up the esophagus.

The present inventors conducted a study on the importance of suppressing nc886 in esophageal squamous cell carcinoma, and propose a method to more accurately predict the prognosis of esophageal squamous cell carcinoma patients.

The present inventors studied the importance of inhibiting nc886 in esophageal squamous cell carcinoma, and confirmed that low expression of nc886 in esophageal squamous cell carcinoma promotes the proliferation of cancer cells. Furthermore, nc886 expression delays the cell cycle, and the cell cycle It was confirmed that the gene was regulated and the target genes of AKT were changed. It is confirmed that the prognosis of esophageal squamous cell carcinoma patients can be predicted by measuring the expression of nc886 in patients with esophageal squamous cell carcinoma, the increase or decrease in the target gene expression of AKT, and the increase or decrease in the expression of genes related to the existing TNM stage and cell cycle. Thus completed the present invention.

As described above, the present invention comprises: a) measuring the expression level of nc886 from a biological sample isolated from a patient with esophageal squamous cell carcinoma; b) measuring the expression level of any one or more of genes whose expression changes according to an increase in nc886 among AKT target genes and genes whose expression changes according to an increase in nc886 among genes related to cell cycle; To provide a method for providing information for predicting the prognosis of patients with esophageal squamous cell carcinoma.

In one embodiment of the invention,

a-1) When the expression level of nc886 is low; b-1) the target gene of AKT has a "expression level of a gene or protein reduced by increasing expression of nc886" higher than a normal control sample; Or “The level of a gene or protein increased by increased expression of nc886” is lower than a normal control sample; And/or b-2) among the genes associated with the cell cycle, "the expression level of a gene or protein reduced by an increase in expression of nc886" is higher than a normal control sample; Alternatively, if the expression level of the gene or protein thereof increased by increasing the expression of nc886 among the genes associated with the cell cycle is lower than the control sample, the prognosis of the esophageal squamous cell carcinoma patient may be determined to be poor.

In the present invention, after step b), collecting the information classified according to TNM for the esophageal squamous cell carcinoma; may further include. According to the TNM, the classification score is calculated from 0 to 4, and if the score is 0, it can be judged to be very good if it is 0, good if it is 1, good if it is 2, bad when it is 3, and very bad if it is 4. .

The present invention, in one preferred embodiment, can provide a method for providing information necessary for predicting the prognosis of esophageal squamous cell carcinoma comprising the following steps.

i) collecting information classified according to the expression level of nc886, the expression level of the target gene of AKT, the expression level of genes related to the cell cycle, and TNM from biological samples isolated from patients with esophageal squamous cell carcinoma;

ii) a-1) If the expression level of nc886 is higher than the normal control sample, 0 point is given; if it is lower than the normal sample control, 1 point is given,

a-2) Among the target genes of AKT, the expression level of a gene or its protein that decreases due to an increase in expression of nc886 is higher than a normal control sample, or a gene or its protein level that increases by an increase in expression of nc886” 1 point is given if it is lower than the normal control sample; Among the target genes of AKT, the expression level of a gene or its protein that decreases due to increased expression of nc886” is lower than the normal control sample, or a gene or its protein level that increases by increased expression of nc886” is higher than a normal control sample. If it's high, give it 0,

a-3) The expression level of a gene or its protein that decreases by increasing the expression of nc886 among genes related to the cell cycle is higher than a normal control sample, or increased by an increase in expression of nc886 among genes related to the cell cycle. If the level of expression of a gene or a protein thereof” is lower than a normal control sample, 1 point is given; The expression level of a gene or its protein that decreases due to an increase in expression of nc886 among genes related to the cell cycle is lower than a normal control sample, or a gene or its increase that increases by an increase in expression of nc886 among genes related to the cell cycle. If the level of protein expression” is higher than the normal control sample, 0 point is given,

If the TNM step is 1 or 2, giving 0 points, and if it is 3 or 4, giving 1 point; And

iii) calculating the score of step ii).

In the present invention, among the target genes of AKT, genes that decrease by increasing expression of nc886 are ACLY, BTK, CD14, CDC42, CDKN1A, CDKN1B, DPYSL2, DPYSL3, EIF4E, FOS, FOXO1, FOXO3, FOXO4, GJA1, GRB10, Any gene selected from the group consisting of GRB2, HSPB1, JUN, MAP3K5, MAPT, MYC, NFKBIA, PABPC1, PAK1, PALLD, PIK3R1, PPP2R1B, PRKAA1, PRKCA, PRKCZ, SHC1, SREBF1, SRF and YWHAH, Genes increased by increased expression of nc886 include ATG4B, BCL2L11, CSNK2A1, CTPS, EIF2AK2, FKBP1A, GADD45A, HRAS, MAPK1, MAPKAP1, MTCP1, PCK2, PPP2CB, PPP2R2A, PRKAA2, PTEN, THEM4, YW, HA It may be any one gene selected from.

In addition, among the genes related to the cell cycle, genes increased by increased expression of nc886 are CDKN2A, RBL1, PPP2R1B, CDKN1A or CDKN2C, and genes decreased by increased expression of nc886 are SKP2, CDKN1B, PPP2CB, UBA52, CDK4, It may be RPS27A, PPP2CA, PPP2R2A or CUL1.

In the present invention, the biological sample isolated from the esophageal squamous cell carcinoma patient may be a formalin-fixed paraffin-embedded (FFPE) sample, fresh tissue or frozen tissue of tissue containing cancer cells of the patient, , But is not limited to this.

In one embodiment of the present invention, the level of the gene is polymerase reaction (PCR), reverse transcriptase reaction (RT-PCR), competitive reverse transcriptase reaction (Competitive RT-PCR), real-time reverse transcriptase reaction (Realtime RT) -PCR), RNase protection assay (RPA; RNase protection assay), Northern blotting, next-generation sequencing (NGS) and DNA microarray analysis Can be measured by

In one embodiment of the present invention, the expression level of the protein is Western blot, ELISA (enzyme linked immunosorbent asay), immunostaining, radioimmunoassay (RIA), radioimmunodiffusion, oruktero One or more methods selected from the group consisting of Ouchterlony immune diffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS and protein chip It can be measured by.

The present invention also provides nc886; And ACLY, BTK, CD14, CDC42, CDKN1A, CDKN1B, DPYSL2, DPYSL3, EIF4E, FOS, FOXO1, FOXO3, FOXO4, GJA1, GRB10, GRB2, HSPB1, JUN, MAP3K5, MAPT, MYC, NFKBA, PANKBD , PIK3R1, PPP2R1B, PRKAA1, PRKCA, PRKCZ, SHC1, SREBF1, SRF, YWHAH, ATG4B, BCL2L11, CSNK2A1, CTPS, EIF2AK2, FKBP1A, GADD45A, HRAS, MAPK1, MAPK2, PAPCPP , THEM4, YWHAE, YWHAG, CDKN2A, CDKN2C, SKP2, UBA52, CDK4, RPS27A, PPP2CA, RBL1 or CUL1 gene, or a composition for predicting esophageal squamous cell cancer prognosis comprising an agent capable of detecting a protein encoded by the gene Can provide.

The present invention also provides nc886; And ACLY, BTK, CD14, CDC42, CDKN1A, CDKN1B, DPYSL2, DPYSL3, EIF4E, FOS, FOXO1, FOXO3, FOXO4, GJA1, GRB10, GRB2, HSPB1, JUN, MAP3K5, MAPT, MYC, NFKBA, PANKBD , PIK3R1, PPP2R1B, PRKAA1, PRKCA, PRKCZ, SHC1, SREBF1, SRF, YWHAH, ATG4B, BCL2L11, CSNK2A1, CTPS, EIF2AK2, FKBP1A, GADD45A, HRAS, MAPK1, MAPK2, PAPCPP , THEM4, YWHAE, YWHAG, CDKN2A, CDKN2C, SKP2, UBA52, CDK4, RPS27A, PPP2CA, RBL1 or CUL1 gene, or a kit for predicting esophageal squamous cell cancer prognosis comprising an agent capable of detecting a protein encoded by the gene Can provide.

The present invention can also provide a biomarker composition for predicting resistance or sensitivity to the anticancer agent palbociclib in esophageal squamous cell carcinoma comprising an agent measuring the mRNA or protein level of the nc886, AKT, CDK4, CDKN2A and CKN2C genes. .

The present invention can also provide a kit for predicting the resistance or sensitivity to the anticancer agent palbociclib in esophageal squamous cell carcinoma comprising the above-described composition.

The present invention can also provide a method for providing information for predicting resistance or sensitivity to the anticancer agent palbociclib in esophageal squamous cell carcinoma comprising the following steps.

a) measuring mRNA or protein levels of the nc886, AKT, CDK4, CDKN2A and CKN2C genes in a biological sample; And

(b) The nc886, CDKN2A and CKN2C gene mRNA or its protein level is lower than the control, AKT and CDK4 gene mRNA or its protein level is higher than the control group, esophageal squamous cell carcinoma susceptibility to anticancer agent palbociclib If it is judged that there is, and the mRNA level of the nc886, CDKN2A and CKN2C genes or its protein level is higher than the control group, and the mRNA level of the AKT and CDK4 genes or the protein level thereof is lower than the control group, the anticancer agent in the esophageal squamous cell carcinoma is used as an anticancer agent Determining that there is resistance to.

In one embodiment of the present invention, the biological sample of step a) is a formalin-fixed paraffin-embedded (FFPE) sample of tissue containing cancer cells of a patient with esophageal squamous cell carcinoma, fresh tissue, and It may be one or more selected from the group consisting of frozen tissue. The mRNA levels of step (a) are polymerase reaction (PCR), reverse transcriptase reaction (RT-PCR), competitive reverse transcriptase reaction (Competitive RT-PCR), real-time reverse transcriptase reaction (Realtime RT-PCR), RNase protection assay (RPA), Northern blotting, next-generation sequencing (NGS), and DNA microarray analysis. have. The protein level of step (a) is western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion, ouchterlony immunodiffusion, rocket (Rocket) from the group consisting of immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complete fixation assay, flow cytometry (FACS) and protein chip assay It may be measured by one or more methods selected.

The present invention measures the expression of genes related to AKT target genes and cell cycles regulated by nc886 and nc886 in patients with esophageal squamous cell carcinoma, and comprehensively scores them by summing the existing TNM stages, thereby accurately predicting the prognosis of patients with esophageal squamous cell carcinoma. It has a predictable effect. In addition, by measuring the mRNA or protein level of the nc886, AKT, CDK4, CDKN2A and CKN2C genes, there is an effect that can predict the resistance or sensitivity to the anticancer agent palbosilib in esophageal squamous cell carcinoma.

1 is a result showing that nc886 inhibits the growth of 293T cells, A is a schematic diagram of the plasmid used in the present invention, B is a RT-PCR result confirming the expression of nc886 in a cell line stably inserted with the plasmid, C is a result showing the proliferation of the cell number of the cells used in the present invention, D is a schematic diagram showing a cell mixing experiment, EF is the result of measuring FACS GFP negative and positive cell number.

2 is a result showing that nc886 slows down the cell cycle, A and B are the results of confirming the cell cycle after staining DNA with propidium iodide (PI).

FIG. 3 shows the results of analyzing the nc886-EXP and nc886-KD array data in an integrated MSigDB (Molecular Signature Database).A shows the Z-scores for the 674 Reactome gene set nc886-EXP (x-axis) and nc886-KD. Schematically plotted between (y-axis) to show the correlation, B is the result of showing the Z-score for 615 TFT (TF target gene set) as a scatterplot as described in panel A, and CD is the Reactome gene A bar graph showing the significantly enhanced TFT Z-scores (>3, based on nc886-EXP) in the set and TFT gene sets.

FIG. 4 shows the results showing that nc886 regulates the expression of cell cycle genes, A is the Venn diagram results of cell cycle genes (40 genes) and genes altered by nc886, and B is 108 ESCC patients Volcano map of 19 nc886/G1-related genes showing statistical correlation with nc886 expression in the cohort, C is the correlation between nc886 expression and a representative cell cycle gene in 108 ESCC patient cohorts, D Is the qRT-PCR result of the gene.

5 is a result of nc886 showing the AKT pathway, A is a heat map result showing RPPA data from nc886-KD of ES1, an ESCC cell line, and B is a Western blot result measuring the expression levels of phosphorylated AKT and total AKT. , C is a GSEA result demonstrating whether the AKT gene set a priori defined in nc886 KD shows statistical agreement in ESCC patients according to nc886 expression.

6 is a result of predicting the prognosis of nc886, AKT and cell cycle genes, A is a result showing that the combination of nc886, AKT signature score, cell cycle signature score, and TNM stage is the strongest predictor of relapse and survival, b TNM stage (stage I and II (0)/stage III and IV (1)), degree of nc886 expression (high (0)/low (1) as median), AKT signature score (low (0)/ as median The calculated sum of high (1)), and cell cycle scores (low (0)/high (1) by median values) is a result of stratification in prognostic scores showing pronounced DSS, RFS and OS in the ESCC patient cohort, C is a summary of this study.

FIG. 7 shows the prognostic and predictive values of nc886, A shows that a number of genes including CDK4 are upregulated in Cluster 2 as a result of the undirected cluster of AKT and cell cycle genes in the ESCC cohort, B is the result of confirming the expression of nc886 in Cluster 1 and Cluster 2, C is the result of confirming the expression of nc886 in the esophageal cell line using qRT-PCR, and D is the weak sensitivity of the 30 EC cell line to Palbosiclip As a result, F is the result of analyzing apoptosis of the esophageal cell line showing the inverse correlation of the expression level of nc886 to the sensitivity to Palbociclib.

8 is a schematic diagram summarizing the present invention.

Hereinafter, the present invention will be described in more detail.

Esophageal cancer is a generic term for cancer that occurs in the esophagus. Esophageal cancer is not one of the most common cancers in Korea, but prognosis is one of the most dangerous cancers. Therefore, measuring a patient's prognosis prediction is one of the most important studies. In the present invention, it was confirmed that low expression of nc886 in esophageal squamous cell carcinoma promotes the proliferation of cancer cells. It was confirmed that the expression of nc886 delayed the cell cycle, regulated the cell cycle gene, and changed the target genes of AKT. The prognosis of esophageal squamous cell carcinoma patients can be predicted by measuring the expression of nc886 in patients with esophageal squamous cell carcinoma, increasing or decreasing the target gene expression of AKT, and increasing or decreasing the expression of genes related to the existing TNM stage and/or cell cycle. The present invention was completed by confirming that.

The present invention comprises the steps of measuring the expression level of nc886 from a biological sample isolated from a patient with esophageal squamous cell carcinoma; b) measuring the expression level of any one or more of genes whose expression changes according to an increase in nc886 among AKT target genes and genes whose expression changes according to an increase in nc886 among genes related to cell cycle; It can provide a method for providing information for predicting the prognosis of patients with esophageal squamous cell carcinoma.

In the present invention, the expression of the measured or collected nc886, the increase or decrease of the target gene expression of AKT, the increase or decrease of the expression of the gene associated with the existing TNM stage and/or cell cycle, is scored, and can be determined by summing it The weight of each expression level and stage can be used to more accurately predict the prognosis of patients with esophageal squamous cell carcinoma.

a-1) When the expression level of nc886 is low;

b-1) the expression level of a gene or its protein that decreases by increasing expression of nc886 among the target genes of AKT is higher than a normal control sample; Or “the level of a gene or protein increased by an increase in expression of nc886” is lower than a normal control sample; And/or

b-2) the expression level of a gene or its protein that decreases by increasing the expression of nc886 among genes related to the cell cycle is higher than a normal control sample; Or if the expression level of the gene or its protein increased by increasing the expression of nc886 among genes related to the cell cycle is lower than the control sample as expected; it can be determined that the prognosis of patients with esophageal squamous cell carcinoma is poor.

In one embodiment of the present invention, after step b), collecting the information classified according to TNM for the esophageal squamous cell carcinoma; may further include. When the stage of the TNM is 3 or 4; it can be determined that the prognosis of the esophageal squamous cell carcinoma patient is poor. Poor prognosis may mean that the disease-specific survival year, recurrence free survival, or overall survival is lower than the good prognosis group. The TNM may refer to the standard of the 7th revision of AJCC as a criterion for dividing the stage of esophageal squamous squamous cancer, but is not limited thereto. The score is calculated from 0 to 4, and if the score is 0, it can be judged as very good, 1 is good, 2 is normal, 3 is bad, and 4 is very bad.

The present invention can also provide a method for providing information necessary for predicting the prognosis of esophageal squamous cell carcinoma comprising the following steps.

i) collecting information classified according to the expression level of nc886, the expression level of the target gene of AKT, the expression level of genes related to the cell cycle, and TNM from biological samples isolated from patients with esophageal squamous cell carcinoma;

ii) a-1) If the expression level of nc886 is higher than the normal control sample, 0 point is given; if it is lower than the normal sample control, 1 point is given,

a-2) Among the target genes of AKT, the expression level of a gene or its protein that decreases due to an increase in expression of nc886 is higher than a normal control sample, or a gene or its protein level that increases by an increase in expression of nc886” 1 point is given if it is lower than the normal control sample; Among the target genes of AKT, the expression level of a gene or its protein that decreases due to increased expression of nc886” is lower than the normal control sample, or a gene or its protein level that increases by increased expression of nc886” is higher than a normal control sample. If it's high, give it 0,

a-3) The expression level of a gene or its protein that decreases by increasing the expression of nc886 among genes related to the cell cycle is higher than a normal control sample, or increased by an increase in expression of nc886 among genes related to the cell cycle. If the level of expression of a gene or a protein thereof” is lower than a control sample as expected, 1 point is given; The expression level of a gene or its protein that decreases due to an increase in expression of nc886 among genes related to the cell cycle is lower than a normal control sample, or a gene or its increase that increases by an increase in expression of nc886 among genes related to the cell cycle. If the level of protein expression” is higher than the control sample as expected, give a score of 0,

If the TNM step is 1 or 2, giving 0 points, and if it is 3 or 4, giving 1 point; And

iii) calculating the score of step ii).

The score is a score for statistical convenience, and it is clear that it can be calculated by adding weights to items to be measured according to the patient's condition and expression level. The weight is, for example, divided by the number (n)/1 of the number of genes or proteins reduced by the increase in expression of nc886 among the target genes of AKT, scores are added for each gene, and the scores are added to obtain the total weight. Can be added.

As a result of examining the cell phenotype according to the expression and silence of nc886, it was confirmed that the cells expressing nc886 grow more slowly than the cells not expressing nc886 (FIG. 1C ). To confirm the above results, cells expressing GFP (293T-GFP/nc886 ⁺ or 293T-GFP) were mixed with the same number as the original 293T cells not expressing GFP, and then the ratio of GFP ⁺ /GFP ^- was observed. Did. As a result, GFP cells were depleted as the co-culture continued, and, importantly, the depletion was 293T-GFP/nc886 ⁺ this was worse than that of 293T-GFP (FIGS. 1E-F). It was confirmed that nc886 ⁺ cells proliferate more slowly than nc886 ^- cells.

According to the results of this study, it is thought that the delayed growth of nc886 ⁺ cells may be extended due to the delay in a specific stage of the cell cycle, not due to the death of cells. To confirm this, cells that grow asynchronously After treating the microtubule inhibitor nocodazole, the cell cycle was analyzed by measuring the DNA content by flow cytometry. Upon nocodazole treatment, cells were synchronized and released from G2/M phase after 6 hours to progress to G1 phase. However, after 12 hours, nc886 ⁻ cells were further advanced into the S phase, whereas in the case of the nc886 ⁺ cells, it was confirmed that the progress toward the S phase was delayed (FIGS. 2A and B ). The above data indicate that nc886 delays the transition from G1 to S, but does not affect the conversion from G2/M to G1. The G1 extended period of nc886 ⁺ cells of nc886 ⁺ cells nc886 ^- indicates that the reason for the slower growth than cells.

Accordingly, the present inventors investigated the gene expression pattern to find out why the cells of nc886 ⁺ have longer G1 groups than the nc886 ^- cells. For this, anti-nc886 (oligonucleotide that targets and inhibits nc886) was transiently transfected on Het-1A cells for 2-3 days, and then gene expression was measured by array. The same experiment was performed in a non-specific anti-oligo (anti-control) to nc886, which was used as a control (the value of anti-nc886/anti-control is expressed as nc886-KD). In addition, the same experiment was performed on 293T-nc886 ⁺ and original 293T (nc886 ^- phosphorus) cells (the value of nc886 ⁺ /nc886 ^- is denoted by nc886-EXP). As a result, in the case of the Reactome gene set, Z-scores for 674 pathways were obtained from nc886-EXP and nc886-KD, and a significant negative correlation was observed when plotted as a scatter plot (FIG. 3A). The nc886-EXP and nc886-KD TFT (transcription factors target gene sets; TFT) Z scores also showed a negative correlation (Fig. 3B). Researchers looked at individual genes to see the pathways or transcription factors (TF) most affected by nc886. In the Reactome set, 9 pathways were significantly activated in nc886-EXP (blue bar in FIG. 3). Note that most of the 9 pathways (8 of 9) are related to the cell cycle, and 3 are directly related to the G1/S phase (FIG. 3C, shown in red brackets). Regarding the up-regulated pathway in the nc886-EXP, all 8 pathways showed a significant negative Z score (<-3) in the nc886-KD. When more than 20 TFT-Z score sets were selected from the nc886-EXP, the most notable were E2F related TFs accounting for more than half (12 of 20) (FIG. 3D).

In the G1 phase subset of the Reactome set, 40 genes were searched for crossing into 6,563 genes, and 19 genes with significant changes in both nc886-KD and nc886-EXP (nc886/G1-related genes, FIG. 4A) were obtained. . The genes were analyzed in a population of 108 ESCC patients with nc886 qRT-PCR values and mRNA sequence data available. The majority of nc886/G1-related genes obtained from cellular data (14 of 19) were found to be significantly associated with nc886 in patient data (Figure 4B). Among them, the researchers selected CDKN2A and CDKN2C. As a result of investigating the expression level in 108 patients with ESCC, CDKN2A and CDKN2C showed changes in the same direction as nc886 (FIG. 4C ). Since SKP2 and CUL1 are known to promote G1-S metastasis by degrading CDK inhibitors, SKP2 and CUL1 were investigated. Expression of these two genes was negatively correlated with nc886 in patient and cell line data (Figure 4B-D). When the above data were comprehensively judged, nc886 regulates the cell cycle gene to delay G1-S metastasis.

Since nc886 was shown to inhibit PKR and Dicer, we investigated whether delayed G1 in nc886-EXP can be explained by both proteins. PKR is a protein involved in cell proliferation, and its representative downstream phenomenon is activation of NF-κB. Therefore, NF-κB was examined in the MSigDB TFT set. PKR induces apoptosis when activated, so it must remain latent in naturally proliferating cells. In this situation, when nc886 was expressed (in nc886-EXP), the additional inhibitory role of nc886 against PKR was hardly observed. Thus, PKR is activated by nc886-KD, but the cell cycle phenotype of nc886-EXP could not be explained. In addition, by studying Dicer, a major enzyme in miRNA biogenesis, a set of miRNA target genes, MSigDB MIR, was investigated. No inhibitory effect of nc886 on miRNA pathway was seen in nc886-EXP.

An attempt was made to identify another protein target of nc886. To solve the above problems, a proteome repertoire was measured using a high-efficiency platform called RPPA. Expression values of 172 proteins were obtained and 23 were found to change at statistically significant levels (FIG. 5A ). When the 23 proteins were examined, it was confirmed that there was a change in protein activity in the AKT pathway. Phosphorylation of AKT1 in hr308 and Ser473 increased, and total AKT1 remained relatively unchanged (FIG. 5A ). nc886-KD increased phosphorylation of substrate 1 (AKT1S1, a.k.a., PRAS40) of AKT1 and Ribosomal Protein S6 (RPS6). AKT1S1 is phosphorylated directly by AKT. To verify RPPA data, AKT phosphorylation was measured in nc886-EXP, which observed the cell phenotype (slow cell proliferation due to delay in G1). Western blot results showed that AKT1 was less phosphorylated in Ser473 than 293T-U6 at 293T-U6:nc886 at 6 and 12 hours after release from the nocodazole suspension (Figure 5B). Overall, the data demonstrated the suppression of AKT by nc886. AKT activation leads to changes in gene expression. Therefore, the effect of nc886 on AKT was further evaluated in gene expression data. Of the 184 genes, 34 increased genes and 19 reduced genes were selected from nc886-KD to apply to the array data for 108 ESCC patients (Table 2 and Table 3). In the GSEA plot, 34 and 19 genes were positively correlated positively with nc886 at statistically significant levels (FIG. 5C ). Concordance of nc886/AKT related genes between nc886-KD data and patient data suggests that nc886 also inhibits the AKT pathway in the tumor formation process in vivo.

The association between nc886, AKT and cell cycle genes is a predictor of survival in ESCC patients. The role of nc886 in inhibiting AKT pathway and cell cycle progression was revealed and nc886 related gene characteristics were identified (34 and 19 nc886/AKT-related genes (FIG. 5); 9 and 5 nc886/G1 related genes (FIG. 4B)) . The prognostic usefulness of these genes was tested in 108 ESCC patients. The AKT signature score was generated by the difference in geometric mean between 34 genes up-regulated and 19 genes down-regulated in nc886-KD. The cell cycle signature score was calculated similarly from 9 genes and 5 genes, each negatively correlated with nc886. Cohorts with this score were differentiated according to each median.

According to C-statistics, the combination of the four factors (expression level of nc886, AKT signature score, cell cycle signature score and TNM stage) was the strongest combination that could predict disease recurrence and patient survival (Figure 6A). TNM stage [stage I and II=0; Stage III and IV=1], degree of nc886 expression [high to medium=0; Low=1], AKT signature score [low to medium=0; High=1], cell cycle score [low to medium=0; The prognosis score was calculated by continuously adding high=1 ]. Stratification into 5 subgroups by prognostic score showed distinct DSS, RFS and OS curves in the 108 ESCC patient population (FIG. 6B ). In both branches, the higher the prognosis score is, the shorter the survival time is. Patients with a score of 4 (TNM Stage III and IV, low nc886, high AKT signature score and high cell cycle score) had the worst survival, but those with a score of 0 had the best survival. The AKT downstream gene and cell cycle gene were found to better predict patient survival when combined with nc886 and TNM staging.

Among the target genes of AKT, genes reduced by nc886 are ACLY, BTK, CD14, CDC42, CDKN1A, CDKN1B, DPYSL2, DPYSL3, EIF4E, FOS, FOXO1, FOXO3, FOXO4, GJA1, GRB10, GRB2, HSPB1, JUN, MUN , MAPT, MYC, NFKBIA, PABPC1, PAK1, PALLD, PIK3R1, PPP2R1B, PRKAA1, PRKCA, PRKCZ, SHC1, SREBF1, SRF and YWHAH are any one of the genes selected, and the gene increased by nc886 ATG4B, BCL2L11, CSNK2A1, CTPS, EIF2AK2, FKBP1A, GADD45A, HRAS, MAPK1, MAPKAP1, MTCP1, PCK2, PPP2CB, PPP2R2A, PRKAA2, PTEN, THEM4, YWHAE and YWHAG. .

Among the genes related to the cell cycle, genes increased by increased expression of nc886 are CDKN2A, RBL1, PPP2R1B, CDKN1A, or CDKN2C, and genes decreased by increased expression of nc886 are SKP2, CDKN1B, PPP2CB, UBA52, CDK4, RPS27A, SKP2, PPP2CA, PPP2R2A or CUL1.

The sample may be at least one selected from the group consisting of formalin-fixed paraffin-embedded (FFPE) samples, fresh tissue, and frozen tissue of tissue containing cancer cells of patients with esophageal squamous cell carcinoma.

The level of the gene is polymerase reaction (PCR), reverse transcriptase reaction (RT-PCR), competitive reverse transcriptase reaction (Competitive RT-PCR), real-time reverse transcriptase reaction (Realtime RT-PCR), RNase protection assay ( RPA; RNase protection assay), Northern blotting, next-generation sequencing (NGS), and DNA microarray analysis.

The expression level of the protein is Western blot, ELISA (enzyme linked immunosorbent asay), immunostaining, radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immune diffusion method, low It may be measured by one or more methods selected from the group consisting of rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, and protein chip.

The term "prognosis" of the present invention means a prediction for a medical ear (eg, long-term viability, disease-free survival, etc.) and includes a positive prognosis (positive prognosis) or a negative prognosis (negative prognosis), wherein the negative prognosis Disease progression or mortality such as recurrence, tumor growth, metastasis, and drug resistance, and a positive prognosis is disease remission, such as no disease, and improvement or stabilization of diseases such as tumor regression It includes. In particular, the poor prognosis in esophageal squamous cell carcinoma in the present invention means that the disease-specific survival year, recurrence free survival or overall survival is low (invention) Figure 6B).

The term "prediction" of the present invention means to guess in advance about the medical ear, and the course of the disease (no disease survival rate, no recurrence survival rate or overall survival period) of a patient diagnosed with esophageal squamous cell carcinoma for the purposes of the present invention. It means guessing in advance.

The present invention also provides nc886; And ACLY, BTK, CD14, CDC42, CDKN1A, CDKN1B, DPYSL2, DPYSL3, EIF4E, FOS, FOXO1, FOXO3, FOXO4, GJA1, GRB10, GRB2, HSPB1, JUN, MAP3K5, MAPT, MYC, NFKBA, PANKBD , PIK3R1, PPP2R1B, PRKAA1, PRKCA, PRKCZ, SHC1, SREBF1, SRF, YWHAH, ATG4B, BCL2L11, CSNK2A1, CTPS, EIF2AK2, FKBP1A, GADD45A, HRAS, MAPK1, MAPK2, PAPCPP , THEM4, YWHAE, YWHAG, CDKN2A, CDKN2C, SKP2, UBA52, CDK4, RPS27A, PPP2CA, RBL1 and/or CUL1 genes, or for predicting esophageal squamous cell carcinoma prognosis comprising an agent capable of detecting a protein encoded by the gene Compositions can be provided.

The present invention also provides nc886; And ACLY, BTK, CD14, CDC42, CDKN1A, CDKN1B, DPYSL2, DPYSL3, EIF4E, FOS, FOXO1, FOXO3, FOXO4, GJA1, GRB10, GRB2, HSPB1, JUN, MAP3K5, MAPT, MYC, NFKBA, PANKBD , PIK3R1, PPP2R1B, PRKAA1, PRKCA, PRKCZ, SHC1, SREBF1, SRF, YWHAH, ATG4B, BCL2L11, CSNK2A1, CTPS, EIF2AK2, FKBP1A, GADD45A, HRAS, MAPK1, MAPK2, PAPCPP , THEM4, YWHAE, YWHAG, CDKN2A, CDKN2C, SKP2, UBA52, CDK4, RPS27A, PPP2CA, RBL1 and/or CUL1 genes, or for predicting esophageal squamous cell carcinoma prognosis comprising an agent capable of detecting a protein encoded by the gene Kits can be provided.

The detectable agent is an antibody, antibody fragment, or aptamer that specifically recognizes the nucleic acid sequence of the gene, its complementary nucleic acid sequence, a fragment of the nucleic acid sequences, or a protein encoded by the nucleic acid sequence. , Avidity (avidity multimer) and / or peptidomimetics (peptidomimetics).

The present invention can also provide a biomarker composition for predicting resistance or sensitivity to the anticancer agent palbociclib in esophageal squamous cell carcinoma comprising an agent measuring the mRNA or protein level of the nc886, AKT, CDK4, CDKN2A and CKN2C genes. .

We performed unsupervised clustering of 108 patients with the gene in order to identify two clusters of “1” and “Cluster 2.” “Cluster 2” showed the expression of most genes, including CDK4. Increased (FIG. 7A) and low expression of nc886 (FIG. 7B) was confirmed.

Palbosiclip is an FDA approved CDK4/6 inhibitor. We investigated whether expression of nc886 associated with CDK4 can predict the response of ESCC cells to Palbociclib. Sensitivity to Palvosilib was investigated in the drug sensitivity genomics ( www.cancerRxgene.org) of the cancer database, the largest public resource providing information on the sensitivity of 1,000 cancer cell lines to over 100 compounds. It was confirmed that nc886 had the lowest IC ₅₀ value suggesting that the silenced ESCC cell line TT (FIG. 7C) was a sensitive drug possessed by Palvosiclip (FIG. 7D). Apoptosis analysis for palbociclib treatment confirmed that TT cells had a higher rate of apoptosis than the nc886 expressing esophageal cell line (FIG. 7E ). When the nc886 and related genes were confirmed through the above results, it was confirmed that the prognosis of treatment for palbosiclip in patients with esophageal squamous cell carcinoma can be predicted.

The present invention can also provide a kit for predicting resistance or sensitivity to the anti-cancer agent Palbosiclip in esophageal squamous cell carcinoma comprising the composition of the above-described composition.

The present invention can also provide a method for providing information for predicting resistance or sensitivity to the anticancer agent palbociclib in esophageal squamous cell carcinoma comprising the following steps.

a) measuring mRNA or protein levels of the nc886, AKT, CDK4, CDKN2A and CKN2C genes in a biological sample; And

(b) The nc886, CDKN2A and CKN2C gene mRNA or its protein level is lower than the control, AKT and CDK4 gene mRNA or its protein level is higher than the control group, esophageal squamous cell carcinoma susceptibility to anticancer agent palbociclib If it is judged that there is, and the mRNA level of the nc886, CDKN2A and CKN2C genes or its protein level is higher than the control group, and the mRNA level of the AKT and CDK4 genes or the protein level thereof is lower than the control group, the anticancer agent in the esophageal squamous cell carcinoma is used as an anticancer agent Determining that there is resistance to.

In the present invention, the biological sample is one selected from the group consisting of formalin-fixed paraffin-embedded (FFPE) samples, fresh tissue, and frozen tissue of tissue containing cancer cells of patients with esophageal squamous cell carcinoma. It may be abnormal. The fresh tissue can be tissue, whole blood, serum or plasma. The mRNA levels of step (a) are polymerase reaction (PCR), reverse transcriptase reaction (RT-PCR), competitive reverse transcriptase reaction (Competitive RT-PCR), real-time reverse transcriptase reaction (Realtime RT-PCR), RNase protection assay (RPA), Northern blotting, next-generation sequencing (NGS), and DNA microarray analysis. have. The protein level of step (a) is western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion, ouchterlony immunodiffusion, rocket (Rocket) from the group consisting of immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complete fixation assay, flow cytometry (FACS) and protein chip assay It may be measured by one or more methods selected.

Hereinafter, examples will be described in detail to help understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

Example 1. Materials

HEK-293T cells (American Type Culture Collection) were cultured at 5% CO ₂ and 37° C. using DMEM medium containing 10% fetal bovine serum and 1% Antibiotic-Antimycotic. The plasmid "pLPCX-U6" was created by inserting the U6 promoter in the pLPCX vector, and the "pCAGGS-GFP" was made by subcloning the green fluorescent protein (GFP) gene into the pCAGGS vector. The nc886 expression plasmids (“pLPCX-U6-pre-886” and “pCAGGS-GFP/nc886”) were inserted into the vectors (“pLPCX-U6” and “pCAGGS-GFP”) (length as simulated in FIG. 1A) (101nt-649nt). Clones isolated by standard plasmid protocol were transformed into HEK-293T cells with Lipofectamine™ 2000 reagent and nc886 ⁺ or nc886 ⁻ cell lines were established. Antibodies to phosphorylation-AKT (Ser 473), AKT and GAPDH were purchased from Cell Signaling Technology (Beverly, MA).

Example 2. Measurement of cell proliferation, cell synchronization and cell cycle analysis

Cell numbers were counted directly on a hemocytometer using a microscope or flow cytometry (FACSC™ II, BD Biosciences). For cell synchronization in the G2/M step, 100 ng/ml nocodazole (nocodazole; Sigma-Aldrich) was treated for 18 hours. After 18 hours, the medium was replaced with nocodazole-free medium. Cells were then collected at 0, 6 and 12 hours to measure DNA content. Cells were fixed with 70% ethanol, DNA was stained with propidium iodide, and DNA content was analyzed using FACSC™ II. FACS data were analyzed using FACS Diva 7 software (BD bioscience; San Jose, CA).

Example 3. RNA isolation and measurement

Total RNA from 293T-derived nc886 ⁺ or control cells was isolated using Trizol reagent (Life Technologies) according to the manufacturer's instructions. DNA was synthesized with amfiRivert kit (GenDEPOT). nc886 was amplified by AccuPower Taq PCR PreMix (BIONEER) and the PCR product was visualized on an agarose gel. MRNAs for CDKN2A, CDKN2C, CUL1 and SKP2 were quantified with qRT-PCR using AccuPower GreenStar™ qPCR MasterMix (BIONEER) and ExicyclerTM 96 Real-Time Quantitative Thermal Block (BIONEER). The primers used for this are shown in Table 1 below.

gene direction Sequence GAPDH Forward "ggccaaggtcatccatgacaactt" Backward "tagaggcagggatgatgttctgga" nc886 Forward "cgggtcggagttagctcaagcgg" Backward "aagggtcagtaagcacccgcg" CDKN2A Forward "ggcagtaaccatgcccgcatagat" Backward "catgcctgcttctacaaacccaca" CDKN2C Forward "aatggatttggaaggactgcgctg" Backward "aaagtgtccaggaaacctgctctg" CUL1 Forward "actcagctgtcctccaggttcaaa" Backward "tgtccttttcaccatccactcgct" SKP2 Forward "cctatcactcagtcggtgctatga" Backward "aacagttgaagggtaccatctggc"

Example 4. ESCC patients

(1) histologically confirmed with ESCC localized to the chest, (2) undergoing complete esophageal resection with adequate lymph node resection, no chemotherapy or radiotherapy before surgery, and (3) adjacent to the paired tumor in the tumor bank. Normal tissue was available (4) and was followed up completely. The following patients were excluded; (1) received pre-surgical chemotherapy and/or radiation therapy in all periods except confirmation of relapse, (2) had a previous cancer diagnosis, (3) double primary concurrent cancer, (4) cervical esophageal cancer, or 5) There was no complete monitoring. Regular follow up was conducted twice a year by phone or mail in April and October. A chest computed tomography (CT) scan was routinely performed during the first follow-up after discharge. In addition, all patients had regular evaluations, such as regular blood tests, chest x-rays, and chest CT scans every 3 months for the first 2 years. All patients were then monitored annually. Positron emission tomography-CT (PET-CT) and esophageal dystrophy were performed annually or more often, if necessary, depending on clinical history and clinical findings. All human specimens were collected with informed patient consent and the study protocol was approved by the Institutional Ethics Committee. After obtaining frozen tissue, total RNA was extracted using the mirVanaTM miRNA Isolation Kit (Ambion, Inc., Austin, TX) according to the manufacturer's instructions.

Example 5. mRNA expression profiling (mRNA array)

Total RNA of 293T-U6 and 293T-U6:nc886 cells was treated with Affymetrix Human Genome U133 Array (Affymetrix, Santa Clara, CA). For nc886 KD in Het-1A cells and ESCC patients, microarray experiments were performed with the HumanHT-12 v4 Expression Beadchip Kit (Illumina, San Diego, CA). According to the manufacturer's protocol, 750 ng of total RNA was labeled with the Total Prep RNA Amplification Kit (Illumina) and hybridized. After scanning the beads chips with a Bead Array Reader (Illumina), the micro-array data was normalized through quantile normalization, and the normalized values were algebraically converted to base 2 scale with R-script. The microarray data is stored in the Gene Expression Omnibus database of the National Center for Biotechnology Information (GSE55856 for ESCC samples and GSE51732 for Het-1A).

Example 6. Reverse phase protein arrays (RPPA)

RPPA data was performed at the Department of System Biology at the MD Anderson Cancer Center. RPPA lysis buffer (1% Triton X-100, 50mM HEPES (pH 7.4), 150mM NaCl, 1.5mM MgCl2, 1mM EGTA, 100mM NaF, 10mM Na pyrophosphate, 1mM Na3VO4, 10% glycerol, 1mM phenylmethylsulfonyl fluoride and 10μg/ml aprotinin) was used. Cell lysates were initially adjusted to 1 μg/μl and diluted 5 fold in lysis buffer to continuously print on nitrocellulose coated slides (Grace Bio-Labs, Bend, OR). Slides were examined with 172 effective antigen antibodies and corresponding secondary antibodies (goat anti-rabbit IgG, goat anti-mouse IgG or rabbit anti-goat IgG). Signals were detected using a diamino benzidine colorimetric reaction and normalized using a supercurve approach.

Example 7. Apoptosis assay in drug treatment

Palbosiclip (PD0332991) HCl (catalog no. S116) was purchased from Selleckchem. 10 ⁶ cells from each cancer cell line were plated in 6-well plates and cultured overnight. Cells were treated with 5 μM of Palboshi Clip and then incubated for 24 hours. Cell suicide analysis was performed by flow cytometry using nnexin V-FITC and 7-AAD (BioLegend. Catalog No. 640922).

Example 8. Statistical analysis

For routine biological assays such as cell counting and qRT-PCR, experiments were performed in three pairs of mean and standard error calculations. The p value was calculated using Student's T-test (one-tailed). Enrichment p-values were calculated by Fisher's exact test. Gene set enrichment analysis (GSEA: http://software.broadinstitute.org/gsea/index.jsp) was performed to determine if the predefined gene set exhibited statistical consistency according to nc886 expression. The correlation between nc886 expression and microarray data was analyzed with Pearson's correlation coefficient. Survival curves were generated by Kaplan-Meier's method, and comparison between groups was performed by log-rank test. Disease specific surviva (DSS) is defined as the time from surgery to death associated with esophageal cancer, and recurrence-free survival (RFS) is the time to death from surgery to confirmed first relapse. It was defined as the time to reach, and the overall survival (OS) was defined as the time from surgery to death. Univariable Cox regression analysis was used to determine if the variable was related to survival. c886 expression, gene marker and TNM (tumor-node-metastasis) stage were used as variables. C statistics from the R language and software environment (http://www.r-project.org) were used to calculate the coincidence index between variables and survival. When p <0.05, it was judged that there was statistical significance, and all tests were conducted in two-tailed. All statistical analyzes were performed with SPSS 24.0 (released at IBM Corp. 2016, IBM SPSS Statistics for Windows version 24.0, Armonk, NY).

Example 9. nc886 inhibits cell proliferation

In previous studies, nc886 was a tumor suppressor in ESCC patient data. Het-1A, a non-malignant esophageal cell line expressing nc886, was subjected to nc886 knockdown (KD) to obtain Het-1A with nc886 knockdown. It was expected that nc886 Het-1A would be more tumorigenic (eg increased cell growth). As expected above, nc886 knockdown-Het-1A expressed several tumor genes. At the same time, however, nc886 KD results in the activation of PKR and the resulting apoptosis, which is consistent with previous studies that nc886 acted as an inhibitor of the pro-apoptotic protein PKR. PKR-mediated apoptosis surpasses all of the other effects of nc886-KD (tumor promoting gene expression) on Het-1A cells, making all further experiments to observe cell phenotype impossible. Therefore, when nc886 was deficient, it was judged that it was not easy to observe the phenotype of the cell, and when nc886 was expressed (gain-of function), it was switched to an approach for observing cells. nc886 is epigenetally silent in ESCC cells (nc886 ^- cells), and by expressing nc886 from several ESCC cell lines, an attempt was made to create an ESCC cell line with the same genetic background as the original cell line except for nc886 expression. . Nevertheless, the nc886 clone could not be isolated. It was assumed that these ESCC cells should be in the nc886 ^- phosphorus state, so that cell proliferation was possible, and thus did not proliferate when artificially produced nc886+ ESCC. Therefore, the human embryonic kidney cell line HEK-293T transformed with the SV40 T antigen (hereinafter referred to as "293T") was used. The use of 293T was chosen as an alternative, but it is a reasonable alternative because the effect of nc886 on gene expression is similar between 293T and Het-1A cells. Two versions of the nc886 293T cell line and corresponding vector control lines (see FIG. 1A for each nomenclature) were made and RT-PCR was measured to confirm nc886 expression (FIG. 1B ). During the cultivation of these cells, it was confirmed that 293T-U6:nc886 and 293T-GFP/nc886 cells grew slower than 293T-U6 and 293T-GFP cells, respectively. Since the increase in cell proliferation capacity is an important phenomenon in tumorigenesis, subsequent studies focused on cell proliferation capacity. Four days after the same cell count was first cultured, the number of 293T-U6 cells was 1.5 times higher than that of 293T-U6:nc886 cells (FIG. 1C ). In addition, a cell mixing experiment was performed using GFP expression in 293T-GFP/nc886 cells. After the same number of mixing with, GFP ⁺ / GFP ^- cells (GFP ⁺⁾ in 293T-GFP / nc886 or 293T-GFP cells the original 293T cells that do not express GFP (GFP) ^- expressing GFP to monitor the rate of Were traced (Figure 1D). GFP cells were depleted as the co-culture continued, and importantly, the depletion was 293T-GFP/nc886 more severe than 293T-GFP (FIGS. 1E-F). When the above data were comprehensively judged, it was confirmed that the cells of nc886 proliferated slowly.

Example 10. nc886 ⁺ cells have a longer G1 duration than nc886 ^- cells

nc886 ⁺ cells have a longer G1 duration than nc886 ^- cells. It was hypothesized that the delayed growth of nc886 ⁺ cells was due to delays at certain stages of the cell cycle. In order to confirm the above hypothesis, cells growing asynchronously were treated with microtubule inhibitor nocodazole and then profiled with a cell cycle using flow cytometry (FIG. 2A ). Upon nocodazole treatment, cells were synchronized at the G2/M step as identified by a single peak at a DNA content of 2n. After 6 hours of nocodazole removal, another peak appeared at the 1n position, and the above results indicate that the cells were released from G2/M and entered the G1 phase. The nc886 ⁺ and nc886 ⁻ cells showed almost the same pattern at this point. However, after 12 hours, it was confirmed that nc886 ⁻ cells further progressed to S (S phage), whereas progression to the S phase was delayed in the case of nc886 ⁺ cells (FIGS. 2A and B ). The above data indicate that nc886 reverses the transition from G1 to S, but does not affect the conversion from G2/M to G1. The extension of the G1 period nc886 ⁺ cell is a cell of nc886 ⁺ nc886 ^- indicates whether more slowly than cell proliferation.

Example 11. nc886 regulates cell cycle genes

Since nc886 has a gene regulation function, the gene expression pattern was examined to find out why the cells of nc886 ⁺ have a longer G1 phase than nc886 ^- cells. Microarrays were performed on the pair of cell lines used in Figures 1 and 2 ("nc886-EXP" compared to 293T-U6, relative values of 293T-U6:nc886). A significant proportion of the genes altered in nc886-EXP would be secondary outcomes. This is because these cells have been maintained for several months until the microarray experiment. Therefore, nc886-EXP data was supplemented with nc886 KD data (“anti-nc886 value compared to control). "The nc886-KD was constructed by transiently transfecting Het-1A cells with anti-nc886 (oligonucleotides targeting and inhibiting nc886) for a few days. Therefore, the nc886-KD data showed that The direct results are better represented: Array data was analyzed for the Molecular Signature Database (MSigDB: http://software.broadinstitute.org/gsea/msigdb) as array data.MSigDB has several sequences based on sequence motifs, pathways, etc. There is a gene collection, for example, a TFT is a collection of target genes for transcription factors (TF), there are 615 sets of TFTs, each set has dozens to hundreds of binding sites for that TF in the promoter region. Contains 4 genes Another example is a set of 674 Reactome sets defined by the Reactome pathway database The overall enrichment or depletion of the set is calculated from the array values of the genes in the set. When it was expressed as a positive or negative z-score, in the case of the Reactome gene set, 674 Z- scores were obtained from nc886-EXP and nc886-KD, and a significant negative correlation was observed compared to the scatterplot (FIG. The TFT Z scores of nc886-EXP and nc886-KD also showed a negative correlation (Fig. 3B).The above results showed that nc886 was similar to the cellular pathway of the esophageal cell line Het-1A in the gene expression and cell pathway of 293T cells. It suggests that it has no effect, suggesting that 293T is suitable for investigating the role of the tumor suppressor factor of nc886 in ESCC Individual genes were examined to see the pathway or TF most affected by nc886. 9 paths in EXP (Z-score Score was cut off >3) was significantly activated (blue bar in FIG. 3). Note that most of the 9 pathways (8 of 9) are somehow related to the cell cycle, and 3 are directly related to the G1/S phase (FIG. 3C, shown in red brackets). Regarding the up-regulated pathway in the nc886-EXP, all 8 pathways showed a significant negative Z score (<-3) in the nc886-KD. When selecting more than 20 TFT-Z score sets from the nc886-EXP, the most notable were E2F related TFs accounting for more than half (12 out of 20) (FIG. 3D). The E2F family TF regulates genes that drive the progression of the G1 vs. S cell cycle. As in Reactome, all 12 TFT sets showed negative Z-scores as opposed to nc886-EXP in nc886-KD. Next, individual cell cycle genes controlled by nc86 were examined. To this end, 40 genes were retrieved from the G1 stage subset of the Reactome set, crossed with 6,563 genes, and 19 genes (nc886/G1 related) with significant (p <0.05) changes in both nc886-KD and nc886-EXP. Gene, FIG. 4A). The genes were analyzed in a population of 108 ESCC patients with nc886 qRT-PCR values and mRNA sequence data available. The majority of nc886/G1-related genes obtained from cellular data (14 of 19) were found to be significantly associated with nc886 in patient data (Figure 4B). Upon closer examination of the 14 genes, we selected CDKN2A (aka, p16INK4a or p14ARF) and CDKN2C for further experiments. This is because it has been reported that inhibiting CDK4/CDK6 restricts cells in G1 and is activated by E2F. As a result of examining the expression level in 108 patients with ESCC, CDKN2A and CDKN2C showed a positive response with nc886 (FIG. 4C ). The expression of CDKN2A and CDKN2C in 293T-U6:nc886 cells increased in line with the long-term persistence of G1 (Figure 2A-B) and enrichment of E2F target genes (Figure 3D) compared to 293T-U6 cells. In addition, SKP2 and CUL1 are known to promote G1-S metastasis by degrading CDK inhibitors, so SKP2 and CUL1 were investigated. These two genes were negatively correlated with nc886 in patient and cell line data (Figure 4B-D). When the above data were comprehensively judged, nc886 regulates the cell cycle gene to delay G1-S metastasis. Since nc886 was shown to inhibit PKR and Dicer, we investigated whether delayed G1 in nc886-EXP could be explained by these two proteins. PKR is a protein involved in cell proliferation and its representative downstream phenomenon is activation of NF-κB. Therefore, NF-κB was examined in the MSigDB TFT set. As described above, PKR induces cell death when activated, so it must remain latent in naturally proliferating cells. In this situation, the inhibitory role of nc886 on PKR has little effect. Although activated by nc886-KD, PKR was unable to explain the cell cycle phenotype of nc886-EXP. In addition, the major enzyme of miRNA biogenesis, Dicer, was studied to investigate the set of miRNA target gene, MSigDB MIR. No inhibitory effect of nc886 on miRNA pathway was observed in nc886-EXP.

Example 12. nc886 inhibits the AKT pathway.

In the previous analysis, PKR and Dicer were unable to explain the role of nc886 in cell cycle progression. Therefore, an attempt was made to identify another protein target of nc886. To solve the above problems, a proteome repertoire was measured using a high-efficiency platform called RPPA. In the above experiment, cell lysates were arranged on nitrocellulose coated slides containing 172 antibodies, and the corresponding protein was comprehensively measured. The antibody contained a protein and its phosphorylated form in a well-known route. RPPA was run on nc886-KD. This is because, as described above, when identifying the direct effect of nc886, short-term KD cells are more advantageous than nc886-EXP. Expression values of 72 proteins were obtained and 23 were found to change at statistically significant levels (FIG. 5A ). When the 23 proteins were examined, it was confirmed that there was a change in the protein in the AKT pathway. The AKT protein consists of three isoforms with strong homology (AKT1, AKT2 and AKT3), and their phosphorylation is active. In the RPPA data, phosphorylation of AKT1 in Thr308 and Ser473 increased, and total AKT1 remained relatively unchanged (FIG. 5A ). In addition, nc886-KD increased phosphorylation of substrate 1 (AKT1S1, a.k.a., PRAS40) of AKT1 and Ribosomal Protein S6 (RPS6). AKT1S1 is phosphorylated directly by AKT. RPS6 is phosphorylated by AKT-promoted Ribosomal Protein S6 Kinase B1 (RPS6KB1, a.k.a., S6K). To verify RPPA data, AKT phosphorylation was measured in nc886-EXP, which observed the cell phenotype (slow cell proliferation due to delay in G1). Western blot results showed that AKT1 was less phosphorylated in Ser473 than 293T-U6 at 293T-U6:nc886 at 6 and 12 hours after release from the nocodazole suspension (FIG. 5B ). Overall, the data demonstrated the suppression of AKT by nc886. AKT activation leads to changes in gene expression. Therefore, the effect of nc886 on AKT was further evaluated in gene expression data. Collection of recent review articles and public databases including Reactome and Biocarta gene sets (http://software.broadinstitute.org/gsea/msigdb/) and RGD (Rat Genome Database; https://rgd.mcw.edu/) A list of 184 AKT downstream genes was selected. Some of these genes are down-regulated while others are down-regulated when AKT is activated. To apply to the array data for 108 ESCC patients, 34 increased genes (Table 2) and 19 reduced genes were selected from nc886-KD among 184 genes (Table 3). In the GSEA plot, 34 and 19 genes were positively correlated positively with nc886 at statistically significant levels (FIG. 5C ). Concordance of nc886/AKT related genes between nc886-KD data and patient data suggests that nc886 also inhibits the AKT pathway in the tumor formation process in vivo.

Example 13. Association between nc886, AKT and cell cycle genes is a predictor of survival in ESCC patients.

The association between nc886, AKT and cell cycle genes is a predictor of survival in ESCC patients. The role of nc886 in inhibiting AKT pathway and cell cycle progression was revealed and nc886 related gene characteristics were identified (34 and 19 nc886/AKT-related genes (FIG. 5); 9 and 5 nc886/G1 related genes (FIG. 4B)) . The prognostic usefulness of these genes was tested in 108 ESCC patients. The AKT signature score was generated by the difference in geometric mean between 34 genes up-regulated and 19 genes down-regulated in nc886-KD. The cell cycle signature score was calculated similarly from 9 genes and 5 genes, each negatively correlated with nc886. Cohorts with this score were differentiated according to each median. TNM staging and nc886 expression were independent prognostic factors for RFS. According to C-statistics, the combination of the four factors (expression level of nc886, AKT signature score, cell cycle signature score and TNM stage) was the strongest combination that could predict disease recurrence and patient survival (FIG. 6A ). TNM stage [stage I and II=0; Stage III and IV=1], nc886 expression level [high to medium value=0; Low=1], AKT signature score [low to medium=0; High=1], cell cycle score [low to medium=0; The prognosis score was calculated by continuously adding high=1 ]. Stratification into 5 subgroups by prognostic score showed distinct DSS, RFS and OS curves in the 108 ESCC patient population (FIG. 6B ). In all three curves, the higher the prognosis score is, the shorter the survival time is. Patients with a score of 4 (TNM Stage III and IV, low nc886, high AKT signature score and high cell cycle score) had the worst survival, but those with a score of 0 had the best survival. AKT downstream genes and cell cycle genes better predicted patient survival when combined with nc886 and TNM staging. To demonstrate how nc886 acts as a tumor suppressor in ESCC, the inhibitory role of nc886 in cell proliferation was demonstrated by identifying AKT and cell cycle genes as downstream events (FIG. 6C ).

Example 14. Therapeutic application to ESCC with undesirable prognosis.

As an inverse approach to demonstrating the association of nc886 with the AKT target gene and cell cycle gene, unsupervised clustering of 108 patients with the gene was performed to identify two distinct clusters named "Cluster1" and "Cluster2" (Fig. 7A). Cluster2 enriched most of the genes, including low expression of CDK4 (Figure 7A) and nc886 (Figure 7B). Some cancer drugs target cell cycle genes. The FDA-approved small molecule CDK4/6 inhibitor, Palbociclib, has been extensively investigated in preclinical studies and clinical trials of a series of human malignancies, including EC. The investigation of nc886, AKT and cell cycle further investigated whether treatment decisions were further improved based on the gene expression profile of the tumor. It was confirmed that the response of ESCC cells to nc886-expressing palbociclib associated with CDK4 can be predicted. Palbosic creep sensitivity was investigated in the drug sensitivity genomics ( www.cancerRxgene.org) of the cancer database, the largest public resource providing information on the sensitivity of nearly 1,000 cancer cell lines to over 100 compounds. It was confirmed that nc886 had the lowest IC ₅₀ value suggesting that the silenced ESCC cell line TT (FIG. 7C) was a sensitive drug possessed by Palvosiclip (FIG. 7D). Apoptosis analysis for palbociclib treatment confirmed that TT cells had a higher rate of apoptosis than the nc886 expressing esophageal cell line (FIG. 7E ). When the nc886 and related genes were confirmed through the above results, it was confirmed that the prognosis of treatment for palbosiclip in patients with esophageal squamous cell carcinoma can be predicted. It was confirmed that the nc886, CDKN2A and CKN2C gene levels were lower than the control group, and the AKT and CDK4 gene levels were higher than the control group.

Claims (19)

a) measuring the expression level of nc886 from a biological sample isolated from a patient with esophageal squamous cell carcinoma; And b) measuring the expression level of any one or more of genes whose expression changes according to an increase in nc886 among the AKT target genes and genes whose expression changes according to an increase in nc886 among genes related to the cell cycle; Including, information providing method for predicting the prognosis of patients with esophageal squamous cell carcinoma. According to claim 1, wherein a-1) when the expression level of the nc886 is low; b-1) the target gene of AKT has a "expression level of a gene or protein reduced by increasing expression of nc886" higher than a normal control sample; Or “The level of a gene or protein increased by increased expression of nc886” is lower than a normal control sample; And/or b-2) Among the genes related to the cell cycle, "the expression level of a gene or its protein that decreases by increasing expression of nc886" is higher than a normal control sample; Or, in the case of the gene associated with the cell cycle, "the expression level of the gene or protein increased by the increase in the expression of nc886" is lower than the control sample as expected; esophageal squamous cell carcinoma of the esophageal squamous cell carcinoma Information provision method for predicting the prognosis of a patient. The method of claim 1, wherein weight is assigned to each expression level measured in steps a) and b) and determined by summing the expression level. The method of claim 1, further comprising: c) in step b) collecting information classified according to TNM for the esophageal squamous cell carcinoma. The method of claim 4, wherein the classification score is calculated from 0 to 4 according to the TNM, and if the score is 0, very good, 1 is good, 2 is normal, 3 is bad, and 4 is very A method of providing information necessary to predict the prognosis of esophageal squamous cell carcinoma, which is judged to be bad. i) collecting information classified according to the expression level of nc886, the expression level of the target gene of AKT, the expression level of genes related to the cell cycle, and TNM from biological samples isolated from patients with esophageal squamous cell carcinoma; ii) a-1) If the expression level of nc886 is higher than the normal control sample, 0 point is given; if it is lower than the normal sample control, 1 point is given, a-2) Among the target genes of the AKT, "the expression level of a gene or its protein that decreases by increasing expression of nc886" is higher than a normal control sample, or "the gene or its protein level that increases by increasing the expression of nc886" "If this is lower than the normal control sample, 1 point is given; Among the target genes of the AKT, "the expression level of the gene or protein reduced by increasing expression of nc886" is lower than the normal control sample, or "the gene or protein level thereof that increases by increasing the expression of nc886" is normal control If it is higher than the sample, 0 point is given; a-3) Among the genes related to the cell cycle, "the expression level of the gene or protein reduced by increasing expression of nc886" is higher than the normal control sample, or the expression of "nc886 is increased in the genes related to the cell cycle." If the expression level of the gene or protein increased by" is lower than the control sample by design, 1 point is given; Among the genes related to the cell cycle, "the expression level of a gene or protein reduced by increasing expression of nc886" is lower than a normal control sample, or a gene that increases by an increase in expression of "nc886" among genes related to the cell cycle Or, if the protein's expression level is higher than the control sample on schedule, give a score of 0; If the TNM step is 1 or 2, giving 0 points, and if it is 3 or 4, giving 1 point; And iii) calculating the score of step ii); Method for providing information for predicting the prognosis of patients with esophageal squamous cell carcinoma comprising a. The method of claim 1 or 6, wherein the gene reduced by nc886 among the target genes of AKT is ACLY, BTK, CD14, CDC42, CDKN1A, CDKN1B, DPYSL2, DPYSL3, EIF4E, FOS, FOXO1, FOXO3, FOXO4, GJA1. , GRB10, GRB2, HSPB1, JUN, MAP3K5, MAPT, MYC, NFKBIA, PABPC1, PAK1, PALLD, PIK3R1, PPP2R1B, PRKAA1, PRKCA, PRKCZ, SHC1, SREBF1, SRF and YWHAH Genes, and the genes increased by nc886 are ATG4B, BCL2L11, CSNK2A1, CTPS, EIF2AK2, FKBP1A, GADD45A, HRAS, MAPK1, MAPKAP1, MTCP1, PCK2, PPP2CB, PPP2R2A, PRKAA2, PTEN, THEM4, YW, consisting of YW Method for providing information for predicting the prognosis of a patient with esophageal squamous cell carcinoma, which is one gene selected from. According to claim 1 or 6, Among the genes associated with the cell cycle, genes increased by increased expression of nc886 are CDKN2A, RBL1, PPP2R1B, CDKN1A or CDKN2C, and genes decreased by increased expression of nc886 are SKP2, A method of providing information for predicting the prognosis of esophageal squamous cell carcinoma patients who are CDKN1B, PPP2CB, UBA52, CDK4, RPS27A, PPP2CA, PPP2R2A or CUL1. The method of claim 1 or 6, wherein the sample is a formalin-fixed paraffin-embedded (FFPE) sample, fresh tissue and frozen tissue of tissue containing cancer cells of a patient with esophageal squamous cell carcinoma. A method for providing information necessary for predicting the prognosis of esophageal squamous cell carcinoma of at least one selected from the group consisting of. The method of claim 1 or 6, wherein the level of the gene is a polymerase reaction (PCR), reverse transcriptase reaction (RT-PCR), competitive reverse transcriptase reaction (Competitive RT-PCR), real-time reverse transcriptase reaction ( Realtime RT-PCR), one or more selected from the group consisting of RNase protection assay (RPA), Northern blotting, next-generation sequencing (NGS) and DNA microarray analysis A method that provides information necessary to predict the prognosis of esophageal squamous cell carcinoma measured by the method. According to claim 1 or 6, The expression level of the protein is Western blot, ELISA (enzyme linked immunosorbent asay), immunostaining (immunostaining), radioimmunoassay (RIA), radioimmunodiffusion, Ou 1 species selected from the group consisting of Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, and protein chip A method for providing information necessary for predicting the prognosis of esophageal squamous cell carcinoma measured by the above method. nc886; And ACLY, BTK, CD14, CDC42, CDKN1A, CDKN1B, DPYSL2, DPYSL3, EIF4E, FOS, FOXO1, FOXO3, FOXO4, GJA1, GRB10, GRB2, HSPB1, JUN, MAP3K5, MAPT, MYC, NFKBIA, PA1, PIK3R1, PPP2R1B, PRKAA1, PRKCA, PRKCZ, SHC1, SREBF1, SRF, YWHAH, ATG4B, BCL2L11, CSNK2A1, CTPS, EIF2AK2, FKBP1A, GADD45A, HRAS, MAPK1, MAPK2, PTC2, MTB2 THEM4, YWHAE, YWHAG, CDKN2A, CDKN2C, SKP2, UBA52, CDK4, RPS27A, PPP2CA, RBL1 or CUL1 genes; Or a composition for predicting the prognosis of esophageal squamous cell carcinoma comprising an agent capable of detecting a protein encoded with the gene. nc886 and; ACLY, BTK,CD14, CDC42, CDKN1A, CDKN1B, DPYSL2, DPYSL3, EIF4E, FOS, FOXO1, FOXO3, FOXO4, GJA1, GRB10, GRB2, HSPB1, JUN, MAP3K5, MAPT, MYC, NFKBIA, PA1, PIK3R1, PPP2R1B, PRKAA1, PRKCA, PRKCZ, SHC1, SREBF1, SRF, YWHAH, ATG4B, BCL2L11, CSNK2A1, CTPS, EIF2AK2, FKBP1A, GADD45A, HRAS, MAPK1, MAPK2, PTC2, MTAP2 THEM4, YWHAE, YWHAG, CDKN2A, CDKN2C, SKP2, UBA52, CDK4, RPS27A, PPP2CA, RBL1 or CUL1 genes; Or a kit for predicting the prognosis of esophageal squamous cell carcinoma comprising an agent capable of detecting a protein encoded with the gene. Biomarker composition for predicting resistance or sensitivity to the anticancer agent palbociclib in esophageal squamous cell carcinoma comprising an agent measuring the mRNA or protein level of the nc886, AKT, CDK4, CDKN2A and CKN2C genes. A kit for predicting resistance or sensitivity to the anticancer agent palbociclib in esophageal squamous cell carcinoma comprising the composition of claim 14. (a) measuring mRNA or protein levels of nc886, AKT, CDK4, CDKN2A and CKN2C genes in a biological sample; And (b) The nc886, CDKN2A and CKN2C gene mRNA or its protein level is low compared to the control, AKT and CDK4 gene mRNA or its protein level is higher than the control group Esophageal squamous cell carcinoma susceptibility to anticancer agent palbociclib If it is judged that there is, and the mRNA level of the nc886, CDKN2A and CKN2C genes or its protein level is higher than the control group, and the mRNA level of the AKT and CDK4 genes or the protein level thereof is lower than the control group, the anticancer agent in the esophageal squamous cell carcinoma is used as an anticancer agent A method of providing information for predicting resistance or sensitivity to the anti-cancer agent Palbosilib in esophageal squamous cell carcinoma, comprising determining that it is resistant to cancer. The method of claim 16, wherein the biological sample of step a) is formalin-fixed paraffin-embedded (FFPE) sample, fresh tissue and frozen tissue of tissue containing cancer cells of the esophageal squamous cell carcinoma patient. A method of providing information for predicting resistance or sensitivity to the anticancer drug Palbosiclip in esophageal squamous cell carcinoma selected from the group consisting of: The method of claim 16, wherein the mRNA level of step (a) is a polymerase reaction (PCR), reverse transcriptase reaction (RT-PCR), competitive reverse transcriptase reaction (Competitive RT-PCR), real-time reverse transcriptase reaction ( Realtime RT-PCR), one or more selected from the group consisting of RNase protection assay (RPA), Northern blotting, next-generation sequencing (NGS) and DNA microarray analysis Method of providing information for predicting resistance or sensitivity to anticancer agent Palbosilib in esophageal squamous cell carcinoma measured by the method The method of claim 16, wherein the protein level of step (a) is Western blotting, Western linked blotting, ELISA (enzyme linked immunosorbent assay), Radioimmunoassay, Radioimmunodiffusion, Ouchterlony ) Immune diffusion method, rocket immunoelectrophoresis, tissue immunostaining method, immunoprecipitation assay, complement fixation assay, flow cytometry (Fluorescence Activated Cell Sorter, FACS) and protein chip Method of providing information for predicting resistance or sensitivity to the anticancer agent Palbosiclip in esophageal squamous cell carcinoma measured by one or more methods selected from the group consisting of analytical methods.

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