CN120758635A

CN120758635A - CircRNA markers for auxiliary diagnosis, prognostic diagnosis or risk stratification of acute myeloid leukemia and their applications

Info

Publication number: CN120758635A
Application number: CN202511276993.XA
Authority: CN
Inventors: 陈莹; 牧启田; 徐耀; 高慧; 李滨权; 徐炜烽
Original assignee: Ningbo Medical Center Li Huili Hospital Affiliated Li Huili Hospital Of Ningbo University; Ningbo Medicalsystem Clinical Laboratories Co ltd; First Affiliated Hospital Of Ningbo University
Current assignee: Ningbo Medical Center Li Huili Hospital Affiliated Li Huili Hospital Of Ningbo University; Ningbo Medicalsystem Clinical Laboratories Co ltd; First Affiliated Hospital Of Ningbo University
Priority date: 2025-09-09
Filing date: 2025-09-09
Publication date: 2025-10-10
Anticipated expiration: 2045-09-09

Abstract

The invention belongs to the technical field of biology, and relates to an auxiliary diagnosis, prognosis diagnosis or risk stratification circRNA marker for acute myelogenous leukemia and application thereof. The invention discovers that the expression level of the circular RNA hsa_circ_0002782 is obviously increased compared with that of a normal marrow specimen, which shows that the circular RNA hsa_circ_0002782 has great potential as an AML biomarker, provides a new and possibly more accurate method for the early detection of AML, and is helpful for timely intervention and treatment. By developing a gene detection product based on circular RNA hsa_circ_0002782, the invention realizes early diagnosis of AML at a molecular level, and the high-precision diagnosis tool can help doctors to identify potential risk patients at the early stage of diseases, so that a more effective personalized medical scheme is provided.

Description

Auxiliary diagnosis, prognosis diagnosis or risk stratification circRNA marker for acute myeloid leukemia and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to an auxiliary diagnosis, prognosis diagnosis or risk stratification circRNA marker for acute myelogenous leukemia and application thereof.

Background

Acute Myeloid Leukemia (AML) is a hematopoietic malignancy characterized by a block of hematopoietic stem and progenitor cell differentiation and clonal proliferation, with a high degree of heterogeneity. Chromosome karyotyping is often used to assess prognosis in AML patients, and with the improvement of the molecular biology technology level, mutation and rearrangement of genes have been widely used clinically. Some molecular mutations such as NPM1, FLT3-ITD, CEBPA, c-KIT, and the like have been incorporated into NCCN and ELN guidelines as markers for AML risk stratification prognostic assessment systems. With the increasing medical level, the vast majority of patients with Acute Promyelocytic Leukemia (APL) can be completely cured by the combined treatment of arsenic trioxide and all-trans retinoic acid due to the discovery of these two drugs.

Despite significant advances in prognostic risk stratification, supportive care, multiple chemotherapeutic drug potentiation, autologous or allogeneic hematopoietic stem cell transplantation (auto/allo-HSCT), etc., prognosis of adult non-AML-M3 patients remains uneconomical due to AML recurrence and refractory characteristics. In recent years, the advent of targeted inhibitors against gene mutant multi-kinases has been promising, but long-term follow-up data is lacking, and therefore there is an urgent need to explore the pathogenesis of AML molecules, find new therapeutic targets, and discover potential markers for AML stratification.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a circular RNA marker for auxiliary diagnosis, prognosis diagnosis or risk stratification of acute myeloid leukemia, so that AML prognosis diagnosis is more accurate and rapid.

The aim of the invention can be achieved by the following technical scheme:

A circular RNA marker for auxiliary diagnosis, prognosis diagnosis or risk stratification of acute myeloid leukemia is RNA hsa_circ_0002782 shown as SEQ ID NO. 1.

The invention also provides application of the detection reagent for the expression level of RNA hsa_circ_0002782 in preparing products for auxiliary diagnosis, prognosis diagnosis or risk stratification of acute myeloid leukemia.

In the above application, the detection reagent for the expression level of RNA hsa_circ_0002782 comprises a primer specifically recognizing RNA hsa_circ_ 0002782.

In the above application, the specific recognition RNA hsa_circ_0002782 primer comprises an upstream primer shown as SEQ ID NO. 2 and a downstream primer shown as SEQ ID NO. 3.

The invention also provides a product for auxiliary diagnosis, prognosis diagnosis or risk stratification of acute myeloid leukemia, which comprises a primer for specifically recognizing RNA hsa_circ_ 0002782.

In the above-mentioned product for auxiliary diagnosis, prognosis or risk stratification of acute myeloid leukemia, the specific primer for recognizing RNA hsa_circ_0003141 comprises an upstream primer shown as SEQ ID NO.2 and a downstream primer shown as SEQ ID NO. 3.

The product for auxiliary diagnosis, prognosis diagnosis or risk stratification of acute myeloid leukemia also comprises a specific recognition internal reference GAPDH primer.

In the above-mentioned product for auxiliary diagnosis, prognosis or risk stratification of acute myeloid leukemia, the specific recognition internal reference GAPDH primer comprises an upstream primer shown as SEQ ID NO. 4 and a downstream primer shown as SEQ ID NO. 5.

The product of the auxiliary diagnosis, prognosis diagnosis or risk stratification of the acute myeloid leukemia is a kit or a gene chip.

The product for auxiliary diagnosis, prognosis diagnosis or risk stratification of the acute myeloid leukemia also comprises pharmaceutically acceptable auxiliary materials.

Compared with the prior art, the invention has the following beneficial effects:

1. The present invention systematically discovers and verifies that the expression level of the circular RNA hsa_circ_0002782 in bone marrow samples of patients with Acute Myeloid Leukemia (AML) is significantly higher than that of normal control groups for the first time. Through high-throughput sequencing, qRT-PCR verification and multi-center clinical sample analysis, the circular RNA is proved to show high specificity and stability up-regulation expression in AML patients, and the expression difference has statistical significance and is not influenced by common reference gene fluctuation. Compared with the traditional diagnosis method relying on morphology and immune typing, the detection means based on the molecular markers has higher sensitivity and specificity. Especially in the early stage of the disease and the stage of the clinical symptoms which are not completely developed, the abnormal expression of hsa_circ_0002782 can be detected, a totally new path is provided for noninvasive or minimally invasive early screening of AML, the clinical goals of early discovery and early intervention are facilitated, and the overall survival rate of patients is remarkably improved.

2. The invention further develops a high-sensitivity gene detection product aiming at hsa_circ_0002782, which can realize the molecular level early diagnosis of AML in a blood or bone marrow sample, and is particularly suitable for the periodic screening of high-risk groups (such as MDS transformed patients and secondary leukemia risk groups after receiving radiotherapy and chemotherapy). The detection method is simple and convenient to operate, has controllable cost and good clinical transformation prospect, and is hopeful to become a supplementary or front screening tool in the AML diagnosis process.

3. The invention uses the circular RNA hsa_circ_0002782 as a biomarker for periodic monitoring, which not only can help evaluate the progress of diseases or treatment response, but also can be used for adjusting an individualized treatment plan. The method improves the efficiency of disease management, ensures more reasonable distribution of medical resources, and reduces the cost of long-term treatment.

Drawings

FIG. 1 shows the result of the first generation sequencing of the product after RT-PCR using hsa_circ_0002782 in example 1.

FIG. 2 shows the amplification efficiency of the reference GAPDH gene and the hsa_circ_0002782 gene of interest established on the basis of AML2 cell line in example 1.

FIG. 3 is a schematic representation of the quantitative RT-PCR assay of example 2 showing up-regulation of hsa_circ_0002782 expression and its diagnostic value in primary AML patients.

FIG. 4 shows the relationship between hsa_circ_0002782 expression and overall survival and event-free survival of acute myeloid leukemia in example 2, where a is overall survival and b is event-free survival.

FIG. 5 shows the results of cell proliferation following knockdown of example 3 in acute myeloid leukemia cell lines AML2 and NB4 using hsa_circ_0002782 small interfering virus, where a is AML2 and b is NB4.

FIG. 6 shows the apoptosis results of example 3 after knockdown of acute myeloid leukemia cell line AML2 using hsa_circ_0002782 small interfering virus.

FIG. 7 shows the apoptosis results of example 3 after knockdown of the acute myeloid leukemia cell line NB4 with hsa_circ_0002782 small interfering virus.

FIG. 8 shows AML CIRCRNA chip screening in example 4, wherein A is hierarchical clustering graph showing the difference of the expression of the circRNA in the AML group and the ALL group and the normal control group, B is scatter graph showing the difference of the expression of the circRNA in the normal control group and the AML group (the outside of two green lines is the difference of more than or equal to 2 times), and C is volcanic graph showing the difference of the expression of the circRNA in the AML group and the normal control group (the red dot is the difference of more than or equal to 2 times).

Fig. 9 is a graph of the diagnostic efficacy of hsa_circ_0002782 in AML as validated by the receiver operating characteristics curve (ROC curve) of example 5.

Detailed Description

The following are specific examples of the present invention, and the technical solutions of the present invention are further described, but the present invention is not limited to these examples.

The present invention relates to a specific circular RNA, namely hsa_circ_0002782, and a primer sequence for specifically identifying a primer pair and an internal reference gene GAPDH.

The sequence and primer composition related by the invention are as follows:

the circular RNA hsa_circ_0002782 shown in SEQ ID NO. 1:

TGTTTAACTGCTGAAGAGATCTTTTCCCTTCATGGCTTTTCAAATGCTACCCAAATAACCAGCTCCAAATTCTCTGTCATCTGTCCAGCAGTCTTACAGCAATTGAACTTTCACCCATGTGAGGATCGGCCCAAGCACAAAACAAGACCAAGTCATTCAGAAGTTTGGGGATATGGATTCCTGTCAGTGACGATTATTAATCTGGCATCTCTCCTCGGATTGATTTTGACTCCACTGATAAAGAAATCTTATTTCCCAAAGATTTTGACCTTTTTTGTGGGGCTGGCTATTGGGACTCTTTTTTCAAATGCAATTTTCCAACTTATTCCAGAGGCATTTGGATTTGATCCCAAAGTCGACAGTTATGTTGAGAAGGCAGTTGCTGTGTTTGGTGGATTTTACCTACTTTTCTTTTTTGAAAGAATGCTAAAGATGTTATTAAAGACATATGGTCAGAATGGTCATACCCACTTTGGAAATGATAACTTTGGTCCTCAAGAAAAAACTCATCAACCTAAAGCATTACCTGCCATCAATGGTGTGACATGCTATGCAAATCCTGCTGTCACAGAAGCTAATGGACATATCCATTTTGATAATGTCAGTGTGGTATCTCTACAG.

In order to effectively detect and quantitatively analyze the existence and the expression level of hsa_circ_0002782, the primer pair for specifically recognizing RNA hsa_circ_0002782 provided by the invention comprises a forward primer shown as SEQ ID No.2 and a reverse primer shown as SEQ ID No. 3:

SEQ ID NO.2:CCTGCTGTCACAGAAGCTAATGG;

SEQ ID NO.3:TGGAGCTGGTTATTTGGGTAGCA;

These two short sequences are designed to pair with the complementary sequence on hsa_circ_0002782, allowing amplification and detection of the circular RNA by molecular biology techniques such as PCR (polymerase chain reaction). The design of the primers takes into account factors such as specificity, efficiency and length of amplified products to ensure optimal experimental results.

In the case of gene expression analysis, a stable reference gene is generally required to normalize data so as to eliminate errors between samples due to a process or individual differences. The upstream primer and the downstream primer of the internal reference GAPDH provided by the invention are shown in SEQ ID NO. 4 as the forward primer and in SEQ ID NO. 5 as the reverse primer:

SEQ ID NO .4:ATGGGGAAGGTGAAGGTCG;

SEQ ID NO .5:GGGTCATTGATGGCAACAATATC。

The pair of primers can be used for amplifying GAPDH MRNA in a sample, so as to correct the relative expression quantity of target RNA.

In the following examples, the present invention detects the expression difference of hsa_circ_0002782 in AML samples by using a real-time fluorescent quantitative PCR method, and analyzes the AML prognosis conditions in the high expression group and the low expression group in combination with clinical information, so that a kit for detecting the gene expression change can be prepared for the AML prognosis diagnosis.

For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. Unless otherwise indicated, the techniques of the present invention are conventional techniques of molecular biology, wherein the enzymes, reagents and reaction conditions involved can be reasonably selected according to the experience of the person skilled in the art, wherein the reagent consumables involved belong to the commercial common products, and the detection means and instruments involved are well known and well known to the person skilled in the art.

Example 1

RT-QPCR and sequencing verification of hsa_circ_0002782 gene and reliability of hsa_circ_0002782 primer

S1, cell collection and pretreatment

And (3) collecting the AML2 cell strain cultured in vitro, washing for 2 times by PBS, adding a reagent (TaKaRa) of 1mL RNAiso Plus into the cell mass, repeatedly blowing and uniformly mixing, and preserving at-80 ℃ for later use.

S2, RNA extraction

200Ul of chloroform was directly added to the RNAiso Plus lysate of 1 above, stirred up and down with force and mixed for 15s, and after 10 minutes of standing, centrifugation was performed at 4℃and 12000g for 15 minutes. The supernatant was taken into a fresh EP tube, added with an equal volume of isopropanol, mixed well, and after 10 minutes of standing, centrifuged at 4℃for 10 minutes at 12000 g. The supernatant was removed, 1ml of 75% ethanol was added and the mixture was washed upside down, centrifuged at 4℃and 12000g for 5 minutes. Removing supernatant, adding 1ml of absolute ethanol, washing upside down, centrifuging at 4deg.C and 12000g for 5min, removing supernatant, and drying at room temperature. And adding a proper volume (20-50 ul) of DEPC water to dissolve RNA according to the amount of RNA precipitation, and measuring the concentration of RNA (A260/A280=1.8-2.1).

S3, cDNA reverse transcription

The cDNA was reverse transcribed using Thermo REVERTAID FIRST STRAND CDNA SYNTHESIS KIT. Reverse transcription reactions were performed according to the following formulations of tables 1 and 2.

TABLE 1 reagents and contents used

The reaction procedure is 65 ℃ for 5 minutes and 4 ℃ for standby;

TABLE 2 reaction system

The reaction procedure was 25 ℃,5 min, 42 ℃,60 min, 70 ℃,5 min, 4 ℃ for the primer;

TABLE 3 primer sequences

S4, quantitative PCR (QPCR)

Hsa_circ_0002782 and GAPDH were amplified using QPCR. This was done using the TaKaRa TB GreenTM Premix Ex TaqTMII (TLI RNASEH Plus) kit. This was accomplished according to the experimental system shown in Table 4 below.

TABLE 4 PCR amplification System

The reaction procedure was a three-step procedure, a standard procedure for PCR amplification, pre-denaturation at 95℃for 1min, a second step of denaturation at 95℃for 15sec, annealing at 60℃for 30sec, and extension at 72℃for 32sec, which was performed for 40 cycles. Melting curve standard procedure 95 ℃,15sec,60 ℃,1min, 95 ℃,15sec,60 ℃,15sec.

S5, agarose electrophoresis

PCR amplification products were detected by 3% agarose gel electrophoresis using GAPDH as an internal reference. The PCR product was sequenced by Shanghai sequencing company and the hsa_circ_0002782 gene was detected by sequence alignment and the result was shown in FIG. 1.

S6, taking cDNA of an AML2 cell strain as a template, vibrating and uniformly mixing PCR amplification products, performing instantaneous centrifugation, gradually diluting according to the modes of 1:100, 1:1000, 1:10000, 1:100000, 1:1000000 and 1:10000000, performing PCR experiments according to the reaction system and the reaction conditions in the fluorescent quantitative PCR to obtain CT values, drawing an amplification efficiency chart, and obtaining the result as shown in FIG. 2, wherein the amplification efficiency of an internal reference primer GAPDH is 99.71%, the amplification efficiency of a target gene hsa_circ_0002782 primer is 98.74%, and the amplification efficiency of the primer is proved to be good.

Example 2

Quantitative RT-PCR detection of hsa_circ_0002782 expression in acute myeloid leukemia bone marrow

S1, collecting and processing bone marrow specimens, namely collecting 190 AML bone marrow specimens of the initial diagnosis of 2016.6-2023.6 months attached to Ningbo university, and normally controlling 21 bone marrow specimens. This study was approved by the first hospital ethics committee affiliated with the university of Ningbo. Bone marrow cell culture flasks were used to harvest bone marrow specimens and patient information was recorded. Mononuclear cells were collected by lymphocyte separation (Ficoll) density gradient centrifugation. Finally adding RNAiso Plus reagent (TaKaRa), repeatedly blowing and mixing uniformly, and preserving at-80 ℃ for later use;

s2, extracting RNA, wherein the method is the same as that of the embodiment 1;

S3, reverse transcription of cDNA, wherein the method is the same as that of the example 1;

S4, quantitative PCR amplification, wherein the method is the same as that of the example 1;

S5, calculating hsa_circ_0002782 gene expression quantity by using a cell strain AML2 cell strain as a control and a 2 ^-ΔΔCt method, wherein delta Ct= [ Ct _{hsa_circ_0002782}-Ct_GAPDH]_{Sample of}-[Ct_{hsa_circ_0002782}-Ct_GAPDH]_{Normal control} ]. Data were statistically analyzed using SPSS 26 software, fisher's exact test was used to compare group differences, and Kaplan-Meier method and Log-rank test were used to analyze group-to-group survival data differences. P <0.05 considered statistically different. From FIG. 3, it is clear that hsa_circ_0002782 is abnormally highly expressed in the initial AML.

S6, analyzing the correlation between hsa_circ_0002782 and clinical characteristics, and the result is shown in Table 5. The results show that there is no statistical significance for the correlation of hsa_circ_0002782 with leukocytes, platelets, bone marrow primordial cells, hemoglobin, etc., but that the complete remission rate of the hsa_circ_0002782 low expression set (74.1%) is significantly higher than that of the hsa_circ_0002782 high expression set (48.7%), indicating that hsa_circ_0002782 functions as an independent indicator in AML and may be prognostic-related.

TABLE 5 expression levels of hsa_circ_0002782 for AML patients versus different clinical indicators

S7, analyzing the relation between hsa_circ_0002782 and AML prognosis, and the result in FIG. 4 shows that the AML with high hsa_circ_0002782 expression has worse overall survival and no event survival, which suggests bad prognosis.

S8, multi-factor analysis of the relation between hsa_circ_0002782 and AML prognosis is carried out, and the results are shown in tables 6 and 7. The results indicate that hsa_circ_0002782 expression can be used as an independent prognostic factor for AML.

TABLE 6 multifactor analysis of overall survival prognosis for AML patients

TABLE 7 multifactor analysis of AML patient event-free survival prognosis

Example 3

In vitro experiments prove the effect of hsa_circ_0002782 on the growth of acute myeloid leukemia cells

S1, synthesizing hsa_circ_0012152 small interfering RNA lentiviral vector (sh-hsa_circ_ 0002782) and irrelevant lentiviral sequences (negative control), wherein the lentiviral vector is marked with puromycin resistance mark, as shown in Table 8. Acute myeloid cell lines AML2 and NB4 were transfected with lentiviral vectors and screened with puromycin to construct a stable sh-hsa_circ_0012152 cell line. The cell growth of the transfected sh-hsa_circ_0012152 cell line was inhibited compared to the cell line transfected with the negative control, and the results are shown in FIG. 5.

TABLE 8 lentiviral sequences

S2, labeling cell lines by using Annexin V-APC/7-AAD, and carrying out apoptosis detection by using an up-flow cytometer, wherein the results are shown in figures 6 and 7, and the apoptosis rate (Q2+Q4) of the sh-hsa_circ_0002782 group is obviously higher than that of the cells of the negative control group.

Example 4

The results of the collection of 3 cases of normal control, 3 cases of primary acute lymphoblastic leukemia and 5 cases of primary acute myeloblastic leukemia bone marrow mononuclear cells for circRNA chip screening showed 255 upregulated (FC. Gtoreq.2, p < 0.05) circRNAs compared with the normal control in FIG. 8, and hsa_circ_0002782 was selected as the target gene according to the abundance and fold of gene expression.

Example 5

Hsa_circ_0002782 expression and diagnostic efficacy in primary AML patients and healthy persons were validated. 100 bone marrow specimens of the first-diagnosis AML patient are taken, 20 bone marrow specimens of the normal control are taken, each sample is treated by adopting the same treatment method as in the embodiment 1, and the hsa_circ_0002782 expression level in each sample is calculated respectively. As shown in the results of FIG. 3, hsa_circ_0002782 was significantly up-regulated in acute myeloid leukemia patients (AML group) compared to healthy people (control group). The diagnostic efficacy in AML was analyzed by the receiver operating specificity curve (ROC curve) (see fig. 9), AUC value of 0.963, sensitivity of 0.90, specificity of 0.95. Analysis results show that hsa_circ_0002782 can be used as a novel molecular marker for AML auxiliary diagnosis.

Based on the results, the invention uses clinical sample verification to determine a new acute myeloid leukemia prognosis gene hsa_circ_0002782, and finds out the relative expression level of hsa_circ_0002782 through multi-factor regression analysis, and hsa_circ_0002782 plays a role in promoting cell growth in acute myeloid leukemia and plays a role of oncogene. Particularly has good predictive value after combining clinical indexes, and can be applied to preparing a reagent or a kit for prognosis diagnosis of patients with acute myelogenous leukemia.

The embodiments herein are not exhaustive of the values in the technical scope of the invention and new technical solutions formed by equivalent substitution of single or multiple technical features in the technical solutions of the embodiments are also within the scope of the invention, and meanwhile, in all the enumerated or non-enumerated embodiments of the present invention, each parameter in the same embodiment only represents an example (i.e. a feasibility solution) of the technical solution, and there is no strict coordination and limiting relationship between each parameter, wherein each parameter can be substituted for each other without violating axiom and the requirement of the invention, except what is specifically stated.

The technical means disclosed by the scheme of the invention is not limited to the technical means disclosed by the technical means, and also comprises the technical scheme formed by any combination of the technical features. While the foregoing is directed to embodiments of the present invention, it will be appreciated by those skilled in the art that changes and modifications may be made without departing from the principles of the invention, and such changes and modifications are intended to be included within the scope of the invention.

The specific embodiments described herein are offered by way of example only to illustrate the spirit of the invention. Those skilled in the art may make various modifications or additions to the described embodiments or substitutions thereof without departing from the spirit of the invention or exceeding the scope of the invention as defined in the accompanying claims.

Claims

1. A circular RNA marker for auxiliary diagnosis, prognostic diagnosis or risk stratification of acute myeloid leukemia, characterized in that the circular RNA marker is RNA hsa_circ_0002782 as shown in SEQ ID NO.1.

2. Application of circular RNA hsa_circ_0002782 expression level detection reagents in the preparation of products for auxiliary diagnosis, prognostic diagnosis or risk stratification of acute myeloid leukemia.

3. The use according to claim 2, wherein the detection reagent for the expression level of circular RNA hsa_circ_0002782 comprises a primer that specifically recognizes circular RNA hsa_circ_0002782.

4. The use according to claim 3, characterized in that the primers that specifically recognize circular RNA hsa_circ_0002782 include the upstream primer shown in SEQ ID NO. 2 and the downstream primer shown in SEQ ID NO. 3.

5. A product for auxiliary diagnosis, prognostic diagnosis, or risk stratification of acute myeloid leukemia, characterized in that it includes primers that specifically recognize circular RNA hsa_circ_0002782.

6. The product for auxiliary diagnosis, prognostic diagnosis, or risk stratification of acute myeloid leukemia according to claim 5, characterized in that the primers that specifically recognize circular RNA hsa_circ_0002782 include the upstream primer shown in SEQ ID NO. 2 and the downstream primer shown in SEQ ID NO. 3.

7. The product for auxiliary diagnosis, prognostic diagnosis or risk stratification of acute myeloid leukemia according to claim 5, further comprising a primer that specifically recognizes an internal reference GAPDH.

8. The product for auxiliary diagnosis, prognostic diagnosis or risk stratification of acute myeloid leukemia according to claim 7, characterized in that the primers specifically recognizing the internal reference GAPDH include the upstream primer shown in SEQ ID NO. 4 and the downstream primer shown in SEQ ID NO. 5.

9. The product for auxiliary diagnosis, prognostic diagnosis or risk stratification of acute myeloid leukemia according to claim 5, characterized in that it is a kit or a gene chip.

10. The product for auxiliary diagnosis, prognostic diagnosis or risk stratification of acute myeloid leukemia according to claim 5, further comprising a pharmaceutically acceptable excipient.