CN111534596A

CN111534596A - Glioma malignant progression and survival prognosis detection molecular marker, temozolomide drug resistance detection target spot and application

Info

Publication number: CN111534596A
Application number: CN202010443721.5A
Authority: CN
Inventors: 吕巧莉; 陈淑慧; 王立冲; 陈俊君; 李敏
Original assignee: Jiangxi Cancer Hospital (jiangxi Cancer Center)
Current assignee: Jiangxi Cancer Hospital (jiangxi Cancer Center)
Priority date: 2020-05-22
Filing date: 2020-05-22
Publication date: 2020-08-14

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a glioma malignant progression and survival prognosis detection molecular marker, a temozolomide drug resistance detection target spot and application. The m6A methylation reader HNRNPC which is highly expressed in glioma tissues and closely related to the occurrence and development process of tumors is screened out by a bioinformatics method, the expression level of the HNRNPC is further analyzed and found to be closely related to the survival rate and prognosis of patients, and the HNRNPC can be used as a molecular marker of glioma malignant progression and an independent survival prognosis factor by verifying a large number of clinical tumor tissue samples. Meanwhile, the HNRNPC is knocked out, so that the drug sensitivity of glioma cells to temozolomide can be improved. The invention provides a new diagnosis, treatment and prognosis prediction target for glioma treatment and a new molecular target for improving temozolomide drug resistance.

Description

Glioma malignant progression and survival prognosis detection molecular marker, temozolomide drug resistance detection target spot and application

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a glioma malignant progression and survival prognosis detection molecular marker, a temozolomide drug resistance detection target spot and application.

Background

Gliomas are the most common primary malignancies of the central nervous system. The incidence is low compared to other malignancies, but due to their rapid proliferation and high infiltration of tumor cells, most patients relapse even after extensive excision of normal brain tissue at the tumor margins. Glioblastoma multiforme is a grade IV tumor with stellate cell differentiation, which is highly aggressive, poorly prognostic, with median survival less than 1 year and extremely high mortality. Therefore, the search for molecular markers related to the occurrence and development of glioma can provide theoretical and basic experimental basis for the treatment of patients.

At present, glioma can be clinically treated by means of surgery, radiotherapy, chemotherapy, gene therapy and the like. Due to the limitation of various conditions, the chemotherapy drugs which are more effective for treating glioma are few and few. Temozolomide is a one-line chemotherapy medicament for treating malignant glioma at present, but a considerable number of patients generate drug resistance to temozolomide, so that the treatment effect is poor. The drug resistance mechanism of temozolomide is reported to be complex in the literature, and methyl guanine-DNA methyltransferase (MGMT) mediated DNA repair is well known. However, studies have shown that glioma cells highly expressing MGMT still exhibit resistance to temozolomide, and clinical use of MGMT inhibitors does not provide a good improvement in patients' insensitivity to temozolomide treatment. This indicates that MGMT is not the only molecular marker mediating temozolomide resistance, and there is a strong need to find other more reliable molecular markers for drug resistance to improve the chemotherapeutic effect of glioma patients.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a glioma malignant progression and survival prognosis detection molecular marker, a temozolomide drug resistance detection target spot and application, and aims to solve part of problems in the prior art or at least alleviate part of problems in the prior art.

The invention is realized by the fact that the molecular marker for malignant progression of glioma is an m6A methylation reading element HNRNPC.

A glioma survival prognosis molecular marker, wherein the molecular marker is m6A methylation reading seed HNRNPC.

The application of the molecular marker in preparing a reagent or a kit for glioma diagnosis or treatment or survival prognosis detection.

Further, the kit comprises a reagent for detecting the HNRNPC gene or an expression product thereof.

Further, the expression product of the HNRNPC gene comprises at least one of RNA and HNRNPC protein.

Further, the kit for detecting the RNA transcription level of HNRNPC comprises RNA reverse transcription reagents gDNAeraser, 5 × gDNA Eraser Buffer and RNsae Free dH₂O, 5 × Primescript Buffer 2, Primescript RT Enzyme Mix, RT primer Mix, fluorescent quantitative PCR reagent TB green, RNase-freeH₂O and a primer designed for HNRNPC gene.

A temozolomide drug resistance detection target spot is m6A methylated reader HNRNPC.

The temozolomide drug-resistance detection target is applied to preparation of a reagent or a kit for detecting the temozolomide drug-resistance level or improving the sensitivity of temozolomide.

Further, the reagent or the kit for improving the sensitivity of temozolomide comprises HNRNPC siRNA.

During the methylation modification of m6A, m6A regulatory enzymes work synergistically to maintain the dynamic balance of intracellular RNA methylation levels, and m6A regulatory enzymes fall into three major classes: methyltransferases (including METTL3, METTL14, WTAP, etc.) exert catalytic RNA methylation modification which can be reversed by demethylase (including FTO, ALKBH5, etc.); meanwhile, the m6A modified RNA is specifically recognized by m6A reading protein (comprising YTH structural domain family, IGF2BP, HNRNPC, eIF3 and the like) in cells and regulates the downstream functions of the RNA. The m6A methylation modification can be widely involved in the physiological and pathological processes of the body and is closely related to the tumorigenesis and development. The application finds that the m6A reader HNRNPC is closely developed with glioma, the technology in the application can obtain better chemotherapy effect for glioma patients, prolong the survival time of glioma patients, improve prognosis and provide corresponding theoretical and experimental basis.

In summary, the advantages and positive effects of the invention are:

the m6A methylation reader HNRNPC which is highly expressed in glioma tissues and closely related to the occurrence and development process of tumors is screened by a bioinformatics method, the expression level of the HNRNPC is further analyzed and found to be closely related to the survival rate and prognosis of patients, and the HNRNPC can be used as a molecular marker of glioma malignant progression and an independent survival prognostic factor through the verification of a large number of clinical tumor tissue samples. Meanwhile, the HNRNPC is knocked out, so that the drug sensitivity of glioma cells to temozolomide can be improved. The invention provides a new diagnosis, treatment and prognosis prediction target for glioma treatment and a new molecular target for improving temozolomide drug resistance.

The invention can prepare corresponding glioma early diagnosis kit, survival prognosis and temozolomide drug resistance detection kit by detecting the HNRNPC level in the tumor tissue of a glioma patient, wherein the kit contains a reagent for quantitatively detecting the RNA transcription level of HNRNPC, a reagent for quantitatively detecting the HNRNPC protein expression level and the like. The survival, prognosis and temozolomide drug resistance of the patient are predicted by detecting whether the expression level of the HNRNPC gene in the glioma tissue of the patient is changed.

Drawings

FIG. 1 is the results of the expression of the m6A methylation regulator in glioma and normal tissues, showing that HNRNPC, WTAP, YTHDF2 and YTHDF1 are significantly upregulated in glioma;

FIG. 2 is a correlation and functional analysis between m6A methylation regulators, and the results show that there is an interaction between the regulators, WTAP and FTO are in negative correlation, and the positive correlation between METTL14 and YTHDC1 is the largest; meanwhile, the m6A regulatory factor function is closely related to the generation and development of glioma;

fig. 3 is a study of the expression and overall survival of the m6A methylation regulator, showing that of the 169 gliomas, 159 clustered in one of the clusters and there was a significant difference between the transcriptional profiles of the two clusters: HNRNPC, WTAP and YTHDF2 expression were significantly up-regulated in cluster 2, whereas ALKBH5, YTHDC2 and FTO were significantly down-regulated, while the survival rate of cluster 2 was higher;

FIG. 4 is a functional annotation of the methylation regulator m6A, showing that genes whose expression is significantly changed are closely associated with tumor development;

FIG. 5 is a graph exploring the relationship of m6A methylation regulators to prognosis showing that high HNRNPC expression correlates with good prognosis;

FIG. 6 is a clinical level analysis of the relationship between HNRNPC expression and glioma malignancy and clinical prognosis, showing that HNRNPC expression is significantly up-regulated in glioma tissues and its expression level is closely related to tumor malignancy; furthermore, as the expression of HNRNPC increases, the better the patient's prognostic level;

FIG. 7 is a graph of the drug sensitivity of glioma cell U251 to temozolomide increased by silencing the m6A reader HNRNPC;

FIG. 8 is a summary of the changes in the expression of m6A methylation regulator in glioma and its potential functions.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

The proteins or fragments thereof involved in the present invention may be naturally purified products, or chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants) using recombinant techniques.

The invention discloses a glioma malignant progression and survival prognosis detection molecular marker, a temozolomide drug resistance detection target spot and application thereof, wherein 116 cases of primary glioma tissue samples, 18 cases of normal brain tissue samples of patients with brain trauma surgeries and related clinical information are obtained from Hunan ya Hospital of the university of China. RT-qPCR, HNRNPC antibody and immunohistochemical experiment and other related reagents are all sold in the market.

The present invention will be further described with reference to the following specific examples. The experimental procedures for which specific conditions are not indicated in the following examples are generally carried out according to standard conditions, as described in the molecular cloning instructions (third edition), or according to the manufacturer's recommendations.

Example 1 screening for differentially expressed m6A regulatory factors in gliomas

1. TCGA and GEPIA database analysis and screening target gene

The method comprises the following steps: and (3) screening the m6A methylation regulatory factors which are differentially expressed by analyzing the expression conditions of the m6A methylation regulatory factors in glioma tissues and normal brain tissues in a TCGA database. And the m6A gene which is screened out to be differentially expressed is verified by using a large number of samples (9,736 cases of glioma and 8,587 cases of normal brain tissue) of the GEPIA database.

As a result: the analysis result shows that 10 genes exist in 13 m6A methylation regulating factors: the expression levels of METTL3, WTAP, KIAA1429, ZC3H13, YTHDC2, YTHDF1, YTHDF2, HNRNPC, FTO and ALKBH5 are significantly different (FIG. 1A, B). The selected differential m6A regulatory factor was further verified using a large number of samples from the GEPIA database, and HNRNPC, WTAP, YTHDF2 and YTHDF1 were found to be significantly upregulated in glioma (fig. 1C).

And (4) conclusion: part of the m6A regulatory factor is aberrantly expressed in gliomas.

2. TCGA and STRING online database analysis of correlation and function between m6A regulatory factors

The method comprises the following steps: the correlation between m6A regulators was analyzed by TCGA and STRING online databases. In addition, GO and KEGG pathways are adopted to analyze the functional action of the methylation factors, and the relation between the methylation factors and the occurrence and development of glioma is explored.

As a result: the interaction between the m6A regulatory factors was found by on-line database analysis of TCGA and STRING (fig. 2A). Further analysis showed that: WTAP and FTO are negatively correlated, while METTL14 and YTHDC1 are maximally correlated positively (fig. 2B). GO and KEGG pathway analysis shows that m6A regulatory factor has wide connection with glioma occurrence and development (figure 2C), and participates in processes such as RNA modification, combination, mRNA processing, m6A methyltransferase complex, DNA dioxygenase reversal alkylation damage, and processing of precursor mRNA signal pathway with cap intron and the like. The results show that the m6A regulatory factor function is closely related to the occurrence and development of glioma.

And (4) conclusion: the m6A modification process is closely related to the development of glioma.

Example 2 differentially expressed m6A regulatory factor in gliomas correlates with patient prognosis of survival

1. Expression of m6A methylation regulator and Total survival Studies

The method comprises the following steps: to explore the relevance of m6A regulatory factors to the prognosis of glioma patients, two new glioma clusters (k ═ 2) were identified based on the expression of all genes associated with the level of m6A methylation, due to the large difference in the number of glioma and normal brain tissue samples in the TCGA database. Transcript profile features were compared between

clusters

1 and 2 by PCA principal component analysis, while patient survival was compared between clusters.

As a result: of the 169 gliomas, 159 clustered in one of the clusters (A-C in FIG. 3). The analysis found that clinical features and m6A regulator expression were different between the two clusters (fig. 3D), and that survival features were different between the two clusters, with significant differences in age, race, gender, IDH, P53 mutation and m6A methylation regulator expression (including HNRNPC, ALKBH5, WTAP, YTHDF2, YTHDC2 and FTO), with significant upregulation of HNRNPC, WTAP and YTHDF2 expression in cluster 2, and significant downregulation of ALKBH5, YTHDC2 and FTO (fig. 3F), with significant differences between the two clusters (fig. 3E) and higher survival in cluster 2 patients (fig. 3G) as found by PCA principal component analysis. Therefore, it is concluded that the expression of HNRNPC, WTAP and YTHDF2 is increased, and the survival rate of glioma patients is increased accordingly.

And (4) conclusion: aberrant expression of some of the m6A regulators correlates with glioma patient survival.

2. Functional annotation of m6A methylation regulators

The method comprises the following steps: genes that changed significantly in cluster 2 were identified using GO and KEGG analysis (| log 2-fold change | >1 and normalized p <0.05) and their functions were annotated.

As a result: the analysis results show that the genes with significantly changed expression in the colony participate in the functions of chemical homeostasis, transmembrane transport, membrane intrinsic components, transmembrane signal receptor activity, cGMP-PKG signal pathway, calcium signal pathway and the like, and the functions are closely related to the development of tumor (FIGS. 4A-D).

And (4) conclusion: it is further demonstrated that the m6A modification process is closely related to the development of glioma.

3. Exploring the relationship of m6A methylation regulon to prognosis

The method comprises the following steps: based on the TCGA database, a LASSO Cox regression algorithm is adopted to establish a m6A methylation regulating factor and glioma prognosis prediction model, and univariate and multivariate Cox regression is adopted to analyze the relation between the m6A regulating factor and the overall survival time of the patient. Based on the risk characteristics of HNRNPC, ZC3H13 and YTHDF2, glioma patients were divided into low risk (n ═ 80) and high risk (n ═ 79) groups and analyzed for association of risk high or low with survival. In addition, the ROC curve is used for verifying the capability of the model for predicting the prognosis of the glioma, and the model is further used for carrying out prognosis analysis. Meanwhile, the GEPIA database is used for analyzing the relationship between the expression level of the m6A regulatory factor in the TCGA and the overall survival rate and disease-free survival rate.

As a result: using the m6A methylation regulator and a model for prognosis of glioma established by LASSO Cox regression algorithm (fig. 5A-B), glioma patients (n ═ 159) were classified into low risk (n ═ 80) and high risk (n ═ 79) groups, and analysis showed that the overall survival of glioma patients in the low risk group was significantly higher than that in the high risk group (p ═ 1.076e-02) (fig. 5C). We subsequently performed regression analysis of differentially expressed m6A regulators in the TCGA database, and the results indicated that HNRNPC is a protective gene closely related to overall survival with a hazard ratio HR of 0.988(p of 0.019) (fig. 5D-E).

To investigate the prognostic predictive value of this model, the relationship between risk score and survival status was further analyzed in this example of the invention and heat maps showing the expression levels of the m6A regulator in different risk groups (FIGS. 5F-G). While there was a clear difference in m6A regulator expression and survival status in clinical features between the two groups (fig. 5H). Furthermore, the ROC curve indicates that the model is a good predictor of glioma prognosis (AUC ═ 0.819) (fig. 5I), from which it can be seen that as HNRNPC expression increases, the patient's risk value decreases, which predicts that the patient may have a better prognosis. Meanwhile, in the embodiment of the invention, the high expression of HNRNPC is found to be related to good prognosis by GEPIA analysis (FIG. 5J). The above results indicate that HNRNPC may be a key gene for predicting prognosis of glioma patients.

And (4) conclusion: the m6A reader HNRNPC expression is closely correlated with the prognosis of glioma patients.

Example 3 clinical level validation of the relationship of the m6A reader HNRNPC to the development of glioma and to the clinical prognosis

1. Tissue sample verification of correlation between HNRNPC expression and glioma malignancy and prognosis

The method comprises the following steps: in order to verify whether HNRNPC plays an important role in the occurrence and development of glioma, the mRNA level of HNRNPC in glioma (n-116) and normal brain tissue (n-28) is firstly detected by RT-qPCR (reverse transcription-quantitative polymerase chain reaction) in the embodiment of the invention, and further verified by Western-blot and immunohistochemical staining. Finally, a correlation analysis was performed between the overall survival of the 63 samples followed and HNRNPC expression.

The glioma HNRNPC diagnosis and detection method disclosed by the embodiment adopts a glioma patient tumor tissue or a normal brain tissue, extracts total RNA according to the operation steps of the instruction, the concentration is 150 and 1000 ng/mu l, reversely transcribes the extracted total RNA into cDNA, and stores the residual RNA at-80 ℃. The specific conditions are as follows:

(1) the reverse transcription reaction was as follows:

(ii) System 1 (removal of genomic DNA)

Reaction conditions are as follows: 42 ℃ for 2min, 4 ℃ and + ∞.

② system 2 (reverse transcription)

Directly adding the system 1 after the reaction into the system 2, and uniformly mixing, wherein the reaction condition is 37 ℃ and 15 min; 5s at 85 ℃; 4 ℃ and + ∞.

(2) Manipulation of reverse transcription products

Used for RT-qPCR analysis, the cDNA is mixed evenly, 2 mul is absorbed to prepare a reaction system as follows:

the primer sequences are as follows.

The reaction conditions adopted a two-step PCR standard amplification procedure:

the first step is as follows: denaturation at 95 ℃ for 30s

The second step is that: PCR reaction at 95 deg.C for 5s and 60 deg.C for 30 s; 40 cycles

The third step: the dissolution curves were 95 ℃ for 10s and 65 ℃ for 5s

Primer concentration was 10. mu.M, GAPDH was used as internal reference, as per 2^-ΔCtThe relative expression amount is calculated. Wherein the primer sequence for detecting the horizontal copy number of the HNRNPC gene is as follows:

F:5′-CCTTACCATCAAACACGATGGC-3′，SEQ ID NO.1；

R:5′-ACTTCGAAAAGATTGCCTCCACA-3′，SEQ ID NO.2；

the primer sequence of GAPDH gene horizontal copy number is as follows:

F:5′-CCCATCACCATCTTCCAGGAG-3′，SEQ ID NO.3；

R:5′-GTTGTCATGGATGACCTTGGC-3′，SEQ ID NO.4；

the gene ID of HNRNPC is 3183.

As a result: RT-qPCR results showed that HNRNPC was up-regulated in glioma tissues (fig. 6A, p <0.01) and HNRNPC expression increased with increasing malignancy of glioma (fig. 6B). In addition, Western-blot and immunohistochemical staining further confirmed the upregulation of HNRNPC protein expression in glioma tissues (FIGS. 6C-D). Finally, high HNRNPC expression was found to correlate with good prognosis by correlation analysis of HNRNPC expression with survival for 63 samples of follow-up (fig. 6E).

And (4) conclusion: the expression level of HNRNPC is closely related to the malignant degree and clinical prognosis of glioma.

2. The combination with the clinical information analysis of patients finds that the high expression of HNRNPC is related to good prognosis

The method comprises the following steps: the role of HNRNPC in glioma progression was analyzed according to patient clinical parameters and HNRNPC expression.

As a result: upregulation of HNRNPC expression levels was clearly associated with glioma tumor grading (p ═ 0.0001) regardless of patient age, sex, and tumor location (table 1).

TABLE 1 correlation of patient clinical information with HNRNPC expression levels

And (4) conclusion: HNRNPC may play an important role in tumor development.

3. Further elucidating the relevance of HNRNPC to the prognosis of patients with glioma

The method comprises the following steps: one-way analysis and multivariate Cox regression analysis showed correlation between clinical staging, HNRNPC expression levels and prognosis.

As a result: single factor analysis showed clinical staging (HR 2.508, 95% CI 1.450-4.338, p 0.001), HNRNPC expression levels (HR 0.510, 95% CI 0.282-0.923, p 0.026) and prognostic correlation. Multivariate Cox regression analysis confirmed that clinical staging (HR 2.688, 95% CI 1.359-5.314, p 0.004) and high expression HNRNPC (HR 0.520, 95% CI 0.283-0.955, p 0.035) are independent prognostic factors associated with glioma patient survival (table 2).

TABLE 2 clinical staging, HNRNPC expression levels and prognostic relevance

The results show that the up-regulation of HNRNPC can be used as a good prognostic marker for patients with glioma.

Example 4 silencing the m6A reader HNRNPC increases drug sensitivity of glioma cells to temozolomide

The method comprises the following steps: after the HNRNPC siRNA is transfected, adding a culture medium containing temozolomide with the concentration of 300 mu M, respectively culturing for 24h, 48h, 72h and 96h by adopting MTS, detecting the cell survival rate, further detecting the influence of the HNRNPC on the drug sensitivity of glioma U251 cells by adopting a flow cytometer, adding a culture medium containing temozolomide with the concentration of 300 mu M after the HNRNPC is silenced, and detecting the apoptosis condition after 72 h.

(1) Transfection procedure

(ii) HNRNPC siRNA sequence

CTCGAAACGTCAGCGTGTA，SEQ ID NO.5；

Operation step (II)

Taking a 6-well plate as an example, glioma cells U251 were plated the day before transfection and cultured in DMEM complete medium containing 10% FBS. Cells were transfected when they grew to 60% long confluence. The old cell culture medium was removed prior to transfection and 1.5ml of complete medium was added to each well after two washes with PBS. The transfection system was then formulated: adding 250 μ l of Opti-MEM into 3 1.5ml EP tubes, respectively adding 5 μ l of HNRNPC siRNA and NC fragment, setting the other group of fragments without interference as Control group, gently mixing, and standing for 5 min; another 3 1.5ml EP tubes were added with 250. mu.l of Opti-MEM, and then added with 5. mu.l of lipo-RNAImax, gently mixed and then left to stand for 5 min. Finally, the two systems are gently mixed evenly, and the mixture is added into each hole after standing for 20 min.

(2) Adding chemicals for treatment

Taking temozolomide powder, adding DMEM (DMEM) containing 10% FBS (FBS) to completely dissolve the temozolomide powder, and preparing liquid medicine with the concentration of 300 mu M. After 6h of transfection, the old cell culture medium was removed, and the culture was continued by adding a complete medium containing temozolomide.

(3) MTS Experimental procedure

The log phase grown cells were plated in 96 well plates, 3 × 10 per well³And (3) operating the cells according to the transfection and medicine adding steps, continuously culturing for 24h, 48h, 72h and 96h after adding medicine, and respectively detecting the cell proliferation condition at each time point by adopting MTS. Old medium was removed and the culture medium was removed according to MTS: working solution is prepared by DMEM (DMEM) at the ratio of 1:9, and 100 mul of working solution is added into each well after the mixture is uniformly mixed. Placing in a cell culture box, incubating at 37 deg.C for 30min, and detecting absorbance at 490nm wavelength with an enzyme-labeling instrument.

(4) Flow cytometry detection of apoptosis

After transfection and dosing according to the steps, when the cells grow to 80-90%, collecting cells by trypsinization, transferring the cells into a 1.5ml EP tube, centrifuging at 3000rpm for 10min, and removing supernatant; after PBS is washed once, 195 mul of Annexin V-FITC binding solution, 5 mul of Annexin V-FITC and 10 mul of propidium iodide are sequentially added into each sample, the mixture is gently mixed, the mixture is dyed for 20min in a dark place at room temperature, and the apoptosis condition of the cells is detected by a flow cytometer.

As a result: the interference efficiency of HNRNPC siRNA is detected by RT-qPCR and Western-blot (figure 7A-B), and the MTS result shows that the survival rate of cells is obviously reduced after the HNRNPC is silenced, namely the drug sensitivity to TMZ is improved (figure 7C). Flow cytometry detection results show that the number of apoptosis of HNRNPC knocked out cells is remarkably increased (FIG. 7D), namely, the drug sensitivity of glioma cells to TMZ is remarkably increased and is consistent with MTS results. The results show that HNRNPC can significantly influence the drug sensitivity of glioma cells to temozolomide.

To summarize: according to the invention, the high expression of the m6A methylation reader HNRNPC in glioma is discovered through bioinformatics analysis, and the high expression is closely related to the occurrence and development of glioma, the survival and the prognosis of patients, which suggests that the HNRNPC can be used as a molecular marker for glioma survival prognosis diagnosis. Further, through analysis of clinical glioma tissue samples, the HNRNPC is remarkably and highly expressed in glioma tissues, and the expression condition of the HNRNPC is closely related to the malignancy degree of tumors. Furthermore, with increased expression of HNRNPC, the better the patient's prognosis level. Research shows that the HNRNPC knock-out can up-regulate some genes related to poor prognosis of tumors, such as MAPK3K3 and MTF2, and can also down-regulate DNJA3 genes related to the overall survival time of breast cancer patients; meanwhile, the prognosis of breast cancer patients with low ROBO1 gene expression is poor. Finally, after HNRNPC is silenced, the sensitivity of glioma U251 cells to temozolomide can be obviously increased, and the apoptosis is obviously increased.

In conclusion, the application finds that HNRNPC has important significance in the glioma development process and the temozolomide drug resistance process, detects whether the level of HNRNPC in a glioma patient is up-regulated, and can be used as an auxiliary means for early diagnosis and prognosis of glioma and temozolomide drug resistance, so that a corresponding glioma early diagnosis kit, survival prognosis and temozolomide drug resistance detection reagent kit can be prepared, and the kit contains reagents for quantitatively detecting the RNA transcription level of HNRNPC, including RNA reverse transcription reagents gDNA Eraser, 5 × gDNA Eraser Buffer and RNsae Free dH₂O, 5 × Primescript Buffer 2, Primescript RT Enzyme Mix, RT primer Mix, fluorescent quantitative PCR reagent TB green, RNsae free H₂O and a primer designed for HNRNPC. The survival period and prognosis of the patient are predicted by detecting whether the expression level of the HNRNPC gene in the glioma tissues of the patient is changed.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principles of the present invention are intended to be included within the scope of the present invention.

Sequence listing

<110> Jiangxi province tumor hospital (Jiangxi province cancer center)

<120> glioma malignant progression and survival prognosis detection molecular marker, temozolomide drug resistance detection target spot and application

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Claims

1. A glioma malignant progression molecular marker, which is m6A methylation reading seed HNRNPC.

2. A glioma survival prognosis molecular marker, wherein the molecular marker is m6A methylation reading seed HNRNPC.

3. Use of the molecular marker of claim 1 or 2 for the preparation of a reagent or kit for the diagnosis or treatment of glioma or for the prognostic detection of survival.

4. Use according to claim 3, characterized in that: the kit comprises a reagent for detecting HNRNPC gene or an expression product thereof.

5. Use according to claim 4, characterized in that: the expression product of the HNRNPC gene comprises at least one of RNA and HNRNPC protein.

6. The use according to claim 5, wherein the kit for detecting RNA transcript level of HNRNPC comprises RNA reverse transcription reagents gDNA Eraser, 5 × gDNA Eraser Buffer and RNsae Free dH₂O, 5 × Primescript Buffer 2, Primescript RT Enzyme Mix, RT primer Mix, fluorescent quantitative PCR reagent TB green, RNase-free H₂O and a primer designed for HNRNPC gene.

7. A temozolomide drug resistance detection target spot is m6A methylated reader HNRNPC.

8. The use of the temozolomide drug resistance assay target of claim 7 in the preparation of a reagent or kit for detecting temozolomide drug resistance level or improving temozolomide sensitivity.

9. Use according to claim 8, characterized in that: the kit comprises a reagent for detecting HNRNPC gene or an expression product thereof.

10. Use according to claim 8, characterized in that: the reagent or the kit for improving the sensitivity of temozolomide comprises HNRNPC siRNA.