CN107236817B - LncRNA ENST00000424523.1 and application of LncRNA ENST00000424523.1 gene in diagnostic and therapeutic drugs - Google Patents
LncRNA ENST00000424523.1 and application of LncRNA ENST00000424523.1 gene in diagnostic and therapeutic drugs Download PDFInfo
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Abstract
The invention discloses an LncRNA ENST00000424523.1 and an application of a gene thereof in diagnosis and treatment medicines, belongs to the technical field of biological medicines, and discloses an LncRNA ENST00000424523.1 and an application of the gene thereof in screening or preparing a chip, a preparation or a kit for clinical diagnosis or prognosis of gastric cancer, an LncRNA ENST00000424523.1 and an application of the gene thereof in screening or preparing the treatment medicines for the gastric cancer, wherein the gene serial number of the LncRNA ENST00000424523.1 is LOC 101927497. The invention has promotion effect on clinical diagnosis and prognosis of gastric cancer and preparation of therapeutic drugs.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to LncRNA ENST00000424523.1 and application of genes thereof in diagnosis and treatment medicines.
Background
Gastric cancer is one of the most common digestive system malignant tumors in the world, and statistics show that 70% of gastric cancer patients belong to developing countries; wherein, nearly half of the gastric cancer patients belong to east Asia countries, especially China. The gastric cancer has complex etiology, and the related factors comprise helicobacter pylori infection, diet, environmental carcinogen exposure, genetic susceptibility and the like; environmental carcinogens are considered to be one of the most important factors leading to the development of gastric cancer.
N-nitroso compounds (NOCs) are strong carcinogenic substances related to gastric cancer, and mainly come from various pickling substances, smoking substances or are formed endogenously in life. N-methyl-N '-nitro-N' -nitrosoguanidine (MNNG), one of the N-nitroso compounds, is a chemical mutagen and carcinogenic agent that are widely recognized in the environment. Although animal models and cell models for inducing gastric cancer by MNNG are constructed at present, the research on gastric cancer is advanced to a certain extent, the gastric cancer mechanism related to MNNG still needs to be further defined.
In the past, the mechanism of chemical carcinogen induced gastric cancer has been studied mainly on coding genes, and recently, with the development of biotechnology such as high-throughput sequencing, the association between non-coding RNA and chemical carcinogen induced gastric cancer has been receiving attention. Long non-coding RNAs (LncRNAs) are a class of non-coding RNAs that are greater than 200 nucleotides in length. LncRNA is an important gene expression regulatory element, can regulate the expression of genes at various levels (epigenetics, transcription, post-transcription and the like), and is closely related to various diseases such as the occurrence, development, apoptosis, migration, adhesion and the like of tumors. According to the role of LncRNA in tumorigenesis, it is currently classified into carcinogenic LncRNA and cancer suppressor LncRNA.
The gastric cancer is a disease problem in the present society, the early clinical diagnosis and prognosis are favorable for the treatment of the gastric cancer, and meanwhile, the treatment drug for the gastric cancer is to be further developed. Therefore, it is necessary to combine LncRNA with gastric cancer to provide a new therapeutic concept for diagnosis, prognosis and treatment of gastric cancer.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides LncRNA ENST00000424523.1 and application of genes thereof in diagnosis and treatment medicines. In order to achieve the purpose, the invention adopts the technical scheme that: LncRNA ENST00000424523.1 and application of genes thereof in screening or preparing chips, preparations or kits for clinical diagnosis or prognosis of gastric cancer, wherein the gene sequence number of LncRNA ENST00000424523.1 is LOC 101927497.
In addition, the invention also provides the LncRNA ENST00000424523.1 and the application of the gene thereof in screening or preparing gastric cancer treatment drugs, wherein the gene sequence number of the LncRNA ENST00000424523.1 is LOC 101927497.
In addition, the invention also provides application of the reagent for detecting the LncRNA ENST00000424523.1 in screening or preparing a chip, a preparation or a kit for clinical diagnosis or prognosis of gastric cancer.
In addition, the present invention also provides a chip, preparation or kit for clinical diagnosis or prognosis of gastric cancer, comprising specific primers for determining the transcription amount of LncRNA ENST00000424523.1, wherein the specific primers comprise DNA sequences as shown in Seq ID No.1 and Seq ID No. 2.
The invention firstly proves that the difference of the transcription of the gene of LncRNA ENST00000424523.1 (LOC101927497) in gastric cancer cells and negative cells exists through high-throughput sequencing RNA-Seq, and prepares a specific primer for detecting LncRNA ENST00000424523.1, which can be used as a marker for clinical diagnosis and prognosis of gastric cancer.
In addition, the invention also provides application of the LncRNA ENST00000424523.1 promoter in screening or preparing gastric cancer treatment medicines.
As an improvement of the above technical scheme, the LncRNA ENST00000424523.1 promoter comprises a plasmid promoting overexpression of the gene LOC101927497, wherein the plasmid contains a DNA sequence of the gene LOC 101927497.
The invention constructs the plasmid of the gene LOC101927497 overexpression, designs and synthesizes siRNA of the gene LOC101927497, and the experiment finds that: in GES-1-T (malignant transformation of human gastric mucosal cells) and MKN-28 (gastric cancer cells), the up-regulation expression of LncRNA ENST00000424523.1 can inhibit the proliferation of gastric cancer cells, and the down-regulation expression of LncRNA ENST00000424523.1 can promote the proliferation of gastric cancer cells.
In addition, the present invention provides a therapeutic agent for gastric cancer, which comprises LncRNA ENST00000424523.1 promoter.
As an improvement of the above technical scheme, the LncRNA ENST00000424523.1 promoter comprises a plasmid promoting overexpression of the gene LOC101927497, wherein the plasmid contains a DNA sequence of the gene LOC 101927497.
As a further improvement of the technical scheme, the treatment medicine also comprises a carrier acceptable to students.
In the above technical solutions, the "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextran, glycerol, ethanol, and the like, and combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, or sodium chloride in the composition. The pharmaceutically acceptable carrier may also contain minor amounts of auxiliary substances which enhance the shelf life or effectiveness of the antibody or antibody portion, such as wetting or emulsifying agents, preservatives or buffers.
In addition, the invention also provides siRNA of the gene LOC101927497, wherein the siRNA is siRNA-1, siRNA-2 or siRNA-3, the siRNA-1 consists of RNA sequences shown as Seq ID NO.3 and Seq ID NO.4, the siRNA-2 consists of RNA sequences shown as Seq ID NO.5 and Seq ID NO.6, and the siRNA-3 consists of RNA sequences shown as Seq ID NO.7 and Seq ID NO. 8.
The invention has the beneficial effects that: the invention provides LncRNA ENST00000424523.1 and application of genes thereof in diagnosis and treatment medicines, firstly, the invention verifies that the transcription of the genes (LOC101927497) of LncRNA ENST00000424523.1 in gastric cancer cells and negative cells has difference through high-throughput sequencing RNA-Seq; specific primers for detecting LncRNA ENST00000424523.1 are prepared, and can be applied to clinical diagnosis and prognosis of gastric cancer; both RNA-Seq data and qRT-PCR data show: LncRNA ENST00000424523.1 is expressed and down-regulated in malignant transformed gastric mucosal cells GES-1-T and gastric cancer cells (MKN-28, MGC-803), the up-regulation expression of LncRNA ENST00000424523.1 can inhibit the proliferation of gastric cancer cells, and the down-regulation expression of LncRNA ENST00000424523.1 can promote the proliferation of gastric cancer cells; therefore, the LncRNA ENST00000424523.1 and the gene can be applied to clinical diagnosis, prognosis and treatment of gastric cancer.
Drawings
FIG. 1 is a graph of the results of high throughput RNA-Seq sequencing of GES-1-T cells and GES-1-N cells; fig. 1A shows LncRNA differentially expressed by two cells, and fig. 1B shows mRNA differentially expressed by two cells; in the figure, the differentially expressed LncRNA and mRNA are marked with circles;
fig. 2 is a statistical diagram verifying differential expression of LncRNA; FIG. 2A shows the expression amounts of LncRNA in GES-1-T cells and GES-1-N cells, EN-452050.1 is an abbreviation for ENST00000452050.1, EN-435695.1 is an abbreviation for ENST00000435695.1, and EN-424523.1 is an abbreviation for ENST 00000424523.1; FIG. 2B shows the relative levels of LncRNA ENST00000424523.1 in GES-1 cells, MKN-28 cells and MGC-803 cells; in the figure, p <0.05, p < 0.01;
FIG. 3 shows the effect of up-regulating the expression of LncRNA ENST00000424523.1 on gastric cancer cell proliferation; FIG. 3A shows the relative expression amounts of LncRNA ENST00000424523.1 in GES-1-T cells and MKN-28 cells introduced with over-expression vector pcDNA-LOC 101927497; FIG. 3B shows that the proliferation of GES-1-T cells and MKN-28 cells introduced into the over-expression vector pcDNA-LOC101927497 is inhibited; in the figure, pcDNA-LncRNA is an over-expression vector of the transferred gene LOC101927497, and pcDNA3.1 is a negative control;
fig. 4 shows the effect of down-regulating LncRNA ENST00000424523.1 expression on gastric cancer cell proliferation; FIG. 4A shows the relative expression levels of LncRNA ENS T00000424523.1 in GES-1-T cells and MKN-28 cells transfected with siRNA; FIG. 4B shows that proliferation of GES-1-T cells and MKN-28 cells transfected with siRNA is promoted; in the figure, siRNA is a negative control.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the following detailed description, accompanying tables and drawings.
Example 1 analysis of high throughput RNA-Seq sequencing results MNNG-treated GES-1-T cells produce differentially expressed LncRNA
The method comprises the following steps: the expression difference of LncRNA and mRNA in two cells is analyzed by a high-throughput RNA-Seq sequencing technology by taking MNNG-induced malignant transformation human gastric mucosal cell GES-1-T (T: transformation) and DMSO-treated negative control cell GES-1-N (N: normal) as experimental objects. The expression amounts of LncRNA in GES-1-T and GES-1-N cells were analyzed, and the differentially expressed LncRNA was determined using a standard of a fold difference of 2-fold or more or 0.5-fold or less in expression and p <0.05, and the differentially expressed mRNA was determined using the same standard.
As a result: as a result of analysis, 17626 LncRNA were detected in GES-1-T cells, and 17916 LncRNA were detected in GES-1-N cells. 253 LncRNAs with expression differences in GES-1-T cells compared to GES-1-N cells, 94 LncRNAs with up-regulation and 159 LncRNAs with down-regulation (as shown in FIG. 1A); meanwhile, 751 mRNAs with expression difference in two cells are found, wherein 244 mRNAs with up-regulated expression and 507 mRNAs with down-regulated expression are shown in figure 1B. As shown in Table 1, the 3 LncRNAs most significantly downregulated in GES-1-T cells are listed.
TABLE 1
Example 2 Low expression of LncRNA ENST00000424523.1 in GES-1-T cells and gastric cancer cells
The method comprises the following steps: to further search for LncRNA that may play a role in MNNG-induced GES-1 cell dyscrasia, 3 LncRNAs with more significant variation differences were selected from LncRNAs with expression differences, and the expression levels of the LncRNAs were detected in GES-1-T cells and GES-1-N cells by qRT-PCR. In addition, we also detected the expression of LncRNA ENST00000424523.1 in gastric cancer cells MKN-28 and MGC-803 by qRT-PCR. LncRNA and internal reference GAPDH primer sequences used in qRT-PCR are shown in Table 2, wherein Forward in Table 2 is a Forward primer, and Reverse is a Reverse primer; the cDNA primer sequences of LncRNA ENST00000424523.1, Forward-AATGCTTGGAATGTGGGAGC (shown as Seq ID No. 1) and Reverse-GGCAGCTTTCAGGGGTTTTA (shown as Seq ID No. 2).
As a result: 3 LncRNAs with more obvious variation differences are analyzed, and from the detection result, the LncRNA ENST00000424523.1 is found to be an LncRNA which has not been researched, wherein the NCBI gene number is LOC 101927497. Compared with GES-1-N cells, the expression of LncRNA ENST00000424523.1 in GES-1-T is obviously reduced (fold change is 0.3974799 +/-0.26, p is less than 0.05) (as shown in FIG. 2A); LncRNA ENST00000424523.1 also showed low expression levels in both MKN-28 and MGC-803 gastric cancer cells (as shown in FIG. 2B). These results show that LncRNA ENST00000424523.1 is expressed and down-regulated in MNNG-induced malignant transformation cell GES-1-T and gastric cancer cell line, and that the gene LOC101927497 is likely to play an important role in MNNG-induced gastric cancer as an anti-cancer gene.
TABLE 2
Example 3 Up-regulation of LncRNA ENST00000424523.1 expression to inhibit gastric cancer cell proliferation
The method comprises the following steps: in order to investigate the function of the gene LOC101927497 in the process of MNNG inducing gastric cancer, the function of the gene LOC101927497 in malignant transformation gastric cancer cells GES-1-T and a gastric cancer cell line MKN28 is studied by constructing an over-expression vector pcDNA-LOC 101927497. GES-1-T and MKN-28 cells were transfected with pcDNA-LOC101927497, respectively, 48 hours later, the overexpression effect of LncRNA ENST00000424523.1 was examined by qRT-PCR method, and the influence of the shown LncRNA ENST00000424523.1 on the proliferation of gastric cancer cells was examined by CCK-8 kit.
As a result: after pcDNA-LOC101927497 transfection, the expression level of LncRNA ENST00000424523.1 is obviously up-regulated in GES-1-T and MKN-28 cells (as shown in FIG. 3A); second, the over-expressed LncRNA ENST00000424523.1 significantly attenuated the proliferation of GES-1-T cells and MKN-28 cells (as shown in FIG. 3B). The results indicate that the up-regulation of the expression level of LncRNA ENST00000424523.1 has a significant inhibitory effect on the proliferation ability of gastric cancer cells.
Example 4 Down-Regulation of LncRNA ENST00000424523.1 expression promotes gastric cancer cell proliferation
The method comprises the following steps: the expression of LncRNA ENST00000424523.1 was interfered with siRNA, and proliferation of GES-1-T cells and MKN-28 cells was examined. 3 specific sirnas were synthesized: siRNA-1, siRNA-2, and siRNA-3 (as shown in Table 3), GES-1-T cells and MKN-28 cells were transfected with 3 siRNAs, respectively, 48 hours later, the expression level of LncRNA ENST00000424523.1 was measured by qRT-PCR to verify the interference effect of the siRNAs, and further CCK-8 kit was used to measure the effect of the LncRNA ENST00000424523.1 on the proliferation of gastric cancer cells, which down-regulated the expression.
As a result: after transfection of 3 siRNAs, the expression level of LncRNA ENST00000424523.1 was significantly decreased in both GES-1-T cells and MKN-28 cells (as shown in FIG. 4A); after the expression of LncRNA ENST00000424523.1 is knocked down, the proliferation capacity of GES-1-T and MKN-28 cells is obviously enhanced, and the MKN-28 cells are obvious (as shown in FIG. 4B).
TABLE 3
Finally, it should be noted that the above embodiments are intended to illustrate the technical solutions of the present invention and not to limit the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
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Claims (4)
- The application of LncRNA ENST00000424523.1 in screening gastric cancer treatment drugs, wherein the gene sequence number of LncRNA ENST00000424523.1 is LOC 101927497.
- 2. Application of the reagent for detecting LncRNA ENST00000424523.1 in preparation of a chip or a kit for clinical diagnosis of gastric cancer.
- 3. Use according to claim 2, characterized in that: the chip or the kit comprises specific primers for determining the transcription amount of LncRNA ENST00000424523.1, wherein the specific primers comprise DNA sequences shown as Seq ID No.1 and Seq ID No. 2.
- The application of the LncRNA ENST00000424523.1 promoter in screening or preparing gastric cancer treatment drugs is characterized in that: the LncRNA ENST00000424523.1 promoter is a plasmid for promoting the over-expression of the gene LOC101927497, and the plasmid contains a DNA sequence of the gene LOC 101927497.
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