CN115873944A - Method for auxiliary diagnosis and prognosis evaluation of glioblastoma based on STK32B gene - Google Patents

Abstract

The invention belongs to the field of biomedical specialty, and particularly relates to a related gene marker for auxiliary diagnosis and prognosis evaluation of glioblastoma and application thereof, wherein the marker is an STK32B gene. The invention describes the function of STK32B gene in glioma, and prepares it into reagent kit for evaluating bad prognosis and effect of radiotherapy and chemotherapy.

Description

Method for auxiliary diagnosis and prognosis evaluation of glioblastoma based on STK32B gene

Technical Field

The invention belongs to the field of biomedical specialty, and particularly relates to a related gene marker for auxiliary diagnosis and prognosis evaluation of glioblastoma multiforme and application thereof.

Background

Primary brain tumors consist of tumors originating in the Central Nervous System (CNS), including different kinds of benign and malignant tumors. Glioblastoma (GBM) is the most common primary malignant brain tumor, accounting for about 57% of all gliomas and 48% of all primary malignant Central Nervous System (CNS) tumors. Despite recent advances in the comprehensive treatment of GBM, including surgical resection, radiation therapy, systemic therapy (chemotherapy, targeted therapy) and supportive therapy, the overall prognosis of GBM patients remains poor with median survival of approximately 15 months. Different molecular subtypes of GBM have different gene changes, so that the treatment response of patients is very different, and the prognosis of patients is influenced finally. In addition, the appearance of symptoms associated with neurological function and the reduction in quality of life can have devastating effects on GBM patients and families. The advent of the age of targeted and immunotherapy of tumors now predicts the great promise of our ability to develop more effective and tolerable therapies to combat this aggressive disease.

The protein kinase, as an important signal messenger of cell life activities, can transfer a gamma phosphate group at the end of ATP to a substrate, thereby completing the work of activating the substrate, activating a signal transduction pathway and the like. Protein kinases are capable of regulating many key biological processes such as cell proliferation, survival and migration etc. and therefore, once their activity is deregulated, they are likely to contribute to the various characteristics of tumours. Members of the AGC protein kinase family are involved in different pathological processes affecting human health including cancer, metabolic disorders, cardiovascular diseases, immune diseases, muscular dystrophy and nervous system diseases. The YANK family is a member of the AGC protein kinase family, and although some genetic evidence suggests that YANK is associated with human disease, the biological role of the YANK subtype remains unclear. There is evidence that STK32C (YANK 3) is highly expressed in bladder tumors and, associated with poor clinical characteristics, down-regulation of STK32C expression inhibits tumor development in vitro and in vivo. Also, the deletion of the genetic region containing STK32B (YANK 2) gene was reported in Ellis-van Creveld syndrome patients, suggesting that it may play a role in brain development. The role of STK32B in GBM is unknown, so there is an urgent need to explore the role of STK32B in GBM and develop markers to assist the diagnosis and treatment of GBM. At present, no report is made on a kit for the STK32B gene in glioma.

Disclosure of Invention

In view of the defects in the prior art, the invention mainly provides a GBM auxiliary diagnosis and prognosis evaluation kit based on an STK32B gene and a using method thereof. We found that there was a significant difference in the RNA expression level of the STK32B gene in low-grade gliomas (LGGs) and Gliomas Blastoma (GBM). Compared with astrocytes, the STK32B gene is significantly up-regulated in GBM primary cells, and therefore, by detecting the expression of the STK32B gene transcript level, the malignancy of glioma can be diagnosed. Meanwhile, depending on the expression of the STK32B gene, the prognosis of GBM patients can be evaluated.

In order to achieve the above object, the present invention provides the following technical solutions.

The invention provides application of a reagent for detecting STK32B gene in preparation of a kit for auxiliary diagnosis and prognosis evaluation of glioblastoma, which is characterized in that the kit comprises a PCR primer pair for amplifying the STK32B gene, wherein the primer pair comprises:

the forward primer sequence is 5' of TGGGCATCACAGCCTATGAGCT-3

The reverse primer sequence is 5 'and 3' of TGGACACGCTCCCTTGAACA-.

Further, the kit also comprises a PCR primer pair for amplifying the housekeeping gene 18S, wherein the primer pair is as follows:

the forward primer sequence is 5' ACCCGTTGAACCCCCATTCGTGA-3

The reverse primer sequence is 5.

Further, the kit also comprises a SYBR Green polymerase chain reaction system, wherein the SYBR Green polymerase chain reaction system comprises a PCR buffer solution, dNTPs, SYBR Green fluorescent dye, enzyme-free water and a fluorescent quantitative sample adding plate.

Further, the kit further comprises an RNA extraction reagent, wherein the RNA extraction reagent comprises Trizol, chloroform, isopropanol, 75% ethanol and RNase free water.

Further, the kit also includes a system for reverse transcription of mRNA into cDNA, the reverse transcription reagent 5X RT master mix: primeScript RTase, RNase Inhibitor, random 6 mers, oligo dT Primer, dNTP Mixture and reaction Buffer; fluorescent quantitative PCR reaction system SYBR Premix Ex TaqTM.

Further, the use method of the kit comprises the following steps:

(1) Treating the obtained fresh tumor tissue by liquid nitrogen, grinding and then extracting RNA;

(2) Reverse transcribing the extracted RNA into corresponding cDNA;

(3) Performing fluorescent quantitative PCR amplification on STK32B and 18S genes by using the reverse transcribed cDNA;

(4) With 18S as an internal reference, ct values were recorded for each reaction and the results were expressed as Δ Ct, where Δ Ct = CtGene-Ct18S.

The invention also provides application of the reagent for detecting the STK32B gene in preparation of a kit for auxiliary diagnosis and prognosis evaluation of glioblastoma, and is characterized in that the kit can quantitatively detect the expression level of the STK32B gene.

The invention also provides application of a reagent for detecting the STK32B gene in preparation of a kit for auxiliary diagnosis and prognosis evaluation of glioblastoma, and is characterized in that the kit for auxiliary diagnosis and prognosis evaluation of glioblastoma takes the STK32B gene as a marker.

Furthermore, the kit for auxiliary diagnosis of glioblastoma is used for detecting the expression level of STK32B gene in tissues by a quantitative PCR technology, and the expression level of the STK32B gene is positively correlated with the glioblastoma incidence. Furthermore, the kit for auxiliary diagnosis of glioblastoma is used for detecting the expression level of STK32B gene in tissues by a quantitative PCR technology, and the degree of the STK32B gene in-cancer expression is in negative correlation with the prognosis survival period of glioblastoma.

Compared with the prior art, the invention has the beneficial effects.

(1) The invention discloses the relation between the STK32B gene and glioma disease progression for the first time, the STK32B gene is expected to become a molecular marker for diagnosing GBM, and a new thought is provided for researching a molecular mechanism of glioma disease progression.

(2) The invention proves that the STK32B gene has obvious difference in LGG and GBM transcription expression level for the first time, compared with LGG tissues, the STK32B gene has obvious up-regulation in GBM tissues, and the malignancy degree of the patient glioma can be judged by detecting the expression condition of the STK32B gene transcription level, so that an auxiliary diagnosis kit for the glioblastoma containing the STK32B gene can be prepared, and the method for diagnosing the glioblastoma by detecting the gene transcription level expression is more sensitive and specific, thereby being beneficial to early diagnosis and treatment of diseases.

(3) The invention firstly proves that the expression level of the STK32B gene in the GBM has obvious correlation with the prognosis of patients, and the survival time of the GBM patients with high STK32B expression is obviously shortened compared with the GBM patients with low STK32B expression. By detecting the expression condition of the STK32B transcription level, the prognosis condition of the GBM patient can be estimated, so that the prognosis evaluation kit for the glioblastoma multiforme containing the STK32B gene is prepared, and the prognosis of the patient can be judged more accurately and objectively.

Drawings

FIG. 1: STK32B gene expression is related to malignant phenotypes such as glioma grade and IDH wild type.

FIG. 2: STK32B gene expression can be used as a diagnostic indicator of GBM.

FIG. 3: the protein expression level coded by the STK32B gene in the GBM neurosphere cells is obviously increased.

FIG. 4: the STK32B gene is indicative of a poor prognosis in GBM patients.

Detailed Description

The following examples are merely illustrative of the present invention and should not be construed as limiting thereof. The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental reagents and materials involved are conventional biochemical reagents and materials unless otherwise specified.

Examples

The STK32B gene has obvious difference between GBM and LGG expression in a database and is a sign of malignant progression of glioma.

The research mainly comprises two database platforms, namely Chinese Glioma Genome Atlas (CGGA) and tumor gene Atlas plan (The Cancer Genome Atlas, TCGA). The CGGA database is a database for analyzing a sample of a patient with glioma of China initiated and established by Beijing Tiantan hospital, is a large-scale database which is specific in China and can reveal the molecular mechanism of glioma occurrence and development of Chinese population, and provides a large amount of basis and clinical relevant support for glioma research of China. CGGA _325 contained 182 low-grade glioma (LGG) samples and 139 GBM samples in total in the CGGA database (http:// www.cgga.org.cn); CGGA _693 contained 443 samples and 249 samples of low-grade glioma (LGG) in total. The transcriptional level results for the STK32B gene are derived from the Illumina Hiseq platform RNA-seq results. The TCGA project was originally composed of The National Cancer Institute (NCI) and The national human genome institute (NHGRI) and developed into a large tumor data research platform with relatively comprehensive data results for glioma as one of its earliest studied tumors. The TCGA RNA-seq dataset consists of 702 samples, of which 529 LGG samples and 173 GBM samples.

As shown in fig. 1A, B, and C, in the CGGA and TCGA RNA-seq databases, the expression level of the STK32B gene was significantly increased in the tumor tissue of GBM patients compared to that of LGG patients (CGGA RNA-seq:P < 0.0001，TCGA RNA-seq：P <0.0001 ); also, studies of the IDH mutation status of GBM patients showed that STK32B was expressed in IDH wild-type patients more than mutant patients (fig. 1D, E, F).

2. Diagnostic ROC curves for LGG and GBM were drawn based on the differential RNA-seq expression of the STK32B gene in CGGA and TCGA glioma tissue samples.

Mapping R for diagnosing GBM based on STK32B expression levels of LGG and GBM of the above CGGA and TCGA databasesOC curve. In the ROC curve evaluation method, when the area value AUC under the ROC curve is greater than 0.5, the closer to 1, the better the diagnostic effect. The AUC value has lower accuracy at 0.5-0.7, certain accuracy at 0.7-0.9, and higher accuracy at more than 0.9. As shown in fig. 2A, B and C, the areas under the ROC curves (AUC values) of STK32B diagnostic GBM are CGGA _325 database:0.678 (in the figure 2A of the drawings,P <0.001 CGGA _693 database:0.633 (in the case of FIG. 2B,P <0.001 TCGA database:0.776 (in the case of FIG. 2C,P <0.001 And thus the STK32B gene can be used as an index for diagnosing GBM.

3. And detecting the differential expression of the STK32B gene in the human normal astrocyte and the glioma primary neurosphere cell.

Collecting tissue samples pathologically diagnosed as glioblastoma multiforme (GBM) at the first hospital affiliated to Chinese medical university, extracting primary neurosphere cells from the tissue samples, extracting proteins of the primary neurosphere cells and normal astrocytes (NHA), and detecting differential expression of the proteins by a Western Blotting method. As a result, the expression level of STK32B gene was significantly increased in GBM neurosphere cells as compared with human astrocytes (NHA) (FIG. 3). The protein level verification shows that the STK32B gene expression level can be well reflected by the RNA detected by the fluorescent quantitative PCR, and the reliability of database conclusion is verified.

4. Survival curves were plotted in the CGGA RNA-seq and TCGA RNA-seq databases as the level of STK32B expression.

In the CGGA RNA-seq database, GBM patients were classified into STK32B high-expression group and STK32B low-expression group according to the order of STK32B expression level from small to large, and survival curves were plotted, and FIG. 4A is a survival curve plotted based on the STK32B mRNA expression level in the CGGA RNA-seq database. P =0.0104 by longrank test analysis, i.e. the prognosis is statistically significant; in the TCGA RNA-seq database, for GBM patients, the patients were classified into high expression group and low expression group of STK32B according to the order of their mRNA expression levels from small to large, and a survival curve was drawn, as shown in fig. 4B, longrank test analyzed to obtain P =0.0108, i.e. the prognosis had statistical significance. Further illustrating that in GBM, the prognosis for patients with high expression of STK32B is significantly worse than for patients with low expression of STK 32B.

5. Preparation of GBM auxiliary diagnosis kit based on STK32B gene.

The glioblastoma multiforme auxiliary diagnosis kit comprises a PCR primer pair for amplifying STK32B genes, a PCR primer pair for amplifying housekeeping genes 18S, a SYBR Green polymerase chain reaction system, an RNA extraction reagent and a system for reverse transcription of mRNA into cDNA.

An STK32B gene-based glioblastoma auxiliary diagnosis and prognosis evaluation kit comprises a PCR primer pair for amplifying an STK32B gene, wherein the primer pair comprises:

the forward primer sequence is 5' of TGGGCATCACAGCCTATGAGCT-3

The reverse primer sequence is 5 'and 3' of TGGACACGCTCCCTTGAACA-.

The kit also comprises a PCR primer pair for amplifying housekeeping gene 18S, wherein the primer pair is as follows:

the forward primer sequence is 5' ACCCGTTGAACCCCCATTCGTGA-3

The reverse primer sequence is 5.

The SYBR Green polymerase chain reaction system comprises a PCR buffer solution, dNTPs, SYBR Green fluorescent dye, RNase free water and a fluorescent quantitative sample adding plate.

The RNA extraction reagent comprises Trizol, chloroform, isopropanol, 75% ethanol and RNase-free water.

The system for reverse transcription into cDNA is 5X RT master mix: primeScript RTase, RNase Inhibitor, random 6 mers, oligo dT Primer, dNTP mix, reaction Buffer.

6. An application method of a glioblastoma multiforme auxiliary diagnosis kit based on an STK32B gene.

The detection process comprises the following specific steps:

1) Grinding fresh tissue to be detected under the action of liquid nitrogen, adding 1mL of Trizol into the broken tissue, repeatedly blowing and beating by using a 1mL pipette gun, incubating at room temperature for 5min, and fully separating the nucleoprotein compound.

2) 200ul of chloroform was added to each tube, the tubes were inverted 10 times, left at room temperature for 3min, centrifuged at 12000rpm for 15min at 4 ℃ and the upper aqueous phase was transferred to a new EP tube (approximately 500 ul).

3) Adding isopropanol (500ul, precooled at 4 deg.C) in the same volume as the supernatant, mixing by inversion, standing at room temperature for 10-20min (15 min on average), centrifuging at 12000rpm at 4 deg.C for 10min, discarding the supernatant to obtain a little white precipitate.

4) Adding 1ml of pre-cooled 75% ethanol (RNase-free water diluted absolute ethanol), slowly adding along the tube wall, shaking up and down gently, and centrifuging at 7500rpm at 4 deg.C for 5min.

5) Removing supernatant, drying at room temperature for 1-2min, adding RNase-free water 50-100ul, and blowing gently with pipette if necessary.

6) The concentration and purity of RNA were determined by Nanodrop. The ratio of OD260/OD280 is 1.80-2.0, which indicates that the purity of RNA meets the experimental requirements.

7) Reverse transcription system:

1 μ gRNA volume: x mu L

5X PrimeScript RT Master Mix: 4μL

RNase free water: 16-X mu L

The total system is as follows: 20 μ L

The reverse transcription conditions were: 15min at 37 ℃, 5S at 85 ℃ and 1min at 4 DEG C

8) Fluorescent quantitative PCR sample adding: the fluorescence quantification plate was placed on ice, 3 replicate wells were made for each sample, and the cDNA (diluted 4-5 times and 2. Mu.L added after dilution) was added.

The fluorescent quantitative PCR system comprises:

SYBR Premix Ex TaqTM: 10μL

upstream primer (10 umol): 1 uL

Downstream primer (10 umol): 1. Mu.L

cDNA template: 2 μ L

RNase free water: 6 μ L

The total system is as follows: 20 μ L

9) After the sample adding is finished, the fluorescent quantitative plate film is covered with the sealing plate carefully by using a PE glove, and the fluorescent quantitative plate film is centrifuged to avoid the generation of bubbles.

10 A setup program: 10min at 95 ℃; a total of 40 cycles + dissolution profile were carried out at 95 ℃ for 10min, 95 ℃ for 5S, 60 ℃ for 10S, and 72 ℃ for 10S.

12 Experimental data analysis: with 18S as an internal reference, ct values were recorded for each reaction, the Ct value being the number of cycles that the fluorescence signal in each reaction tube went through to reach a set threshold. Δ Ct = CtGene-Ct18S, and a smaller Δ Ct indicates a higher initial copy number and a higher expression, and Δ Ct is used as an expression level of STK32B for evaluation.

Various other modifications and changes may be made by those skilled in the art based on the above-described technical solutions and concepts, and all such modifications and changes should fall within the scope of the claims of the present invention.

Sequence listing

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Claims (10)

1. The application of a reagent for detecting STK32B gene in the preparation of a glioblastoma multiforme aided diagnosis and prognosis evaluation kit is characterized in that the kit comprises a PCR primer pair for amplifying the STK32B gene, and the primer pair is as follows:

the forward primer sequence is 5' of TGGGCATCACAGCCTATGAGCT-3

The reverse primer sequence is 5 'tggacacgctccacctctgaac-3'.

2. The use of the reagent for detecting STK32B gene in the preparation of the kit for auxiliary diagnosis and prognosis evaluation of glioblastoma according to claim 1, wherein the kit further comprises a PCR primer pair for amplifying housekeeping gene 18S, and the primer pair is as follows:

the forward primer sequence is 5' ACCCGTTGAACCCCCATTCGTGA-3

The reverse primer sequence is 5.

3. The application of the reagent for detecting STK32B gene in the preparation of the kit for auxiliary diagnosis and prognosis evaluation of glioblastoma according to claim 1, wherein the kit further comprises a SYBR Green polymerase chain reaction system, and the SYBR Green polymerase chain reaction system comprises PCR buffer, dNTPs, SYBR Green fluorescent dye, enzyme-free water and a fluorescent quantitative sample adding plate.

4. The use of the reagent for detecting STK32B gene in the preparation of the kit for auxiliary diagnosis and prognosis evaluation of glioblastoma according to claim 1, wherein the kit further comprises an RNA extraction reagent, and the RNA extraction reagent comprises Trizol, chloroform, isopropanol, 75% ethanol and RNase free water.

5. The use of the reagent for detecting STK32B gene in the preparation of the kit for the aided diagnosis and prognosis evaluation of glioblastoma according to claim 1, wherein the kit further comprises a system for reverse transcription of mRNA into cDNA, and the reverse transcription reagent 5X RT master mix: primeScript RTase, RNase Inhibitor, random 6 mers, oligo dT Primer, dNTP mix, reaction Buffer; fluorescent quantitative PCR reaction system SYBR Premix Ex TaqTM.

6. The use of the reagent for detecting STK32B gene in the preparation of the kit for auxiliary diagnosis and prognosis evaluation of glioblastoma according to claim 1, wherein the use method of the kit comprises the following steps:

(1) Treating the obtained fresh tumor tissue by liquid nitrogen, grinding and then extracting RNA;

(2) Reverse transcribing the extracted RNA into corresponding cDNA;

(3) Performing fluorescent quantitative PCR amplification on STK32B and 18S genes by using the cDNA subjected to reverse transcription;

(4) With 18S as an internal reference, ct values were recorded for each reaction and the results were expressed as Δ Ct, where Δ Ct = CtGene-Ct18S.

7. The application of a reagent for detecting STK32B gene in the preparation of a kit for auxiliary diagnosis and prognosis evaluation of glioblastoma, which is characterized in that the kit can quantitatively detect the expression level of the STK32B gene.

8. The application of the reagent for detecting the STK32B gene in preparing the kit for auxiliary diagnosis and prognosis evaluation of glioblastoma is characterized in that the kit for auxiliary diagnosis and prognosis evaluation of glioblastoma takes the STK32B gene as a marker.

9. The use of claim 8, wherein the kit for auxiliary diagnosis of glioblastoma multiforme is used to detect the expression level of STK32B gene in tissue by quantitative PCR technique, and the expression level of STK32B gene is positively correlated to the glioblastoma multiforme incidence.

10. The use of claim 8, wherein the kit for auxiliary diagnosis of glioblastoma is used to detect the expression level of STK32B gene in tissue by quantitative PCR, and the degree of intratumoral expression of STK32B gene is inversely correlated with the prognostic survival of glioblastoma.

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