CN109182528B - Glioblastoma multiforme auxiliary diagnosis and prognosis evaluation kit based on ITGB5 gene and use method thereof - Google Patents

Abstract

The invention belongs to the field of biomedical specialty, and particularly relates to an ITGB5 gene-based glioblastoma multiforme auxiliary diagnosis and prognosis evaluation kit and a using method thereof. The invention describes the functional action of ITGB5 gene in glioma, and prepares a kit for evaluating adverse prognosis and the effect of radiotherapy and chemotherapy.

Description

Glioblastoma multiforme auxiliary diagnosis and prognosis evaluation kit based on ITGB5 gene and use method thereof

Technical Field

The invention belongs to the field of biomedical specialty, and particularly relates to an ITGB5 gene-based glioblastoma multiforme auxiliary diagnosis and prognosis evaluation kit and a using method thereof.

Background

Glioblastoma (GBM) is the most prominent primary malignancy in the Central Nervous System (CNS) of adults. Even with maximal surgical resection, radiation therapy, and adjuvant temozolomide chemotherapy, median survival of GBM patients is only 12-15 months, with only about 10% of GBM patients surviving for more than 5 years. In recent years, the research on the immune microenvironment of tumors has been increasing, and immunotherapy has become a new strategy for solid tumors and has drastically changed the treatment in some specific types of cancer. For example, immune checkpoint inhibitors such as nivolumab and ipilimumab have enabled long-term survival in a subset of patients with advanced melanoma, which were previously incurable with median survival of about 8 months. Disruption of the immune response is a major feature of GBM, particularly the mesenchymal subtype. Tumor-mediated immunosuppression can prevent the eradication of GBM cells and promote tumor progression. Cytokines such as Interleukin (IL) -10, IL-6 and colony stimulating factor 1 (CSF-1) secreted by tumor cells and tumor-associated non-tumor cells play an important role in the above process.

Integrins are heterodimeric cell surface receptors, consisting of 18 α -subunits and 8 β -subunits, which modulate a variety of biological functions in cancer, including proliferation, adhesion, migration and invasion; and simultaneously, the compound is involved in regulating tumor immune microenvironment, angiogenesis and the like. The various integrins involved in disease progression and their association with oncogenes make them attractive targets for tumor therapy. ITGB5 is only associated with ITG α V, and by multiple studies is associated with a variety of pathological conditions. ITGB5 inhibits exogenous apoptosis by enhancing cell survival through coordination with endothelial growth factor receptor 2 (VEGF 2); ITGB5 was found to be involved in angiogenesis in a variety of tumors. Increasing evidence supports the key role of ITGB5 in promoting cancer cell migration, invasion and transforming growth factor beta (TGF- β) -induced epithelial-mesenchymal transition (EMT). However, the role of ITGB5 in gliomas is not known, and therefore, it is urgently needed to explore the role of ITGB5 in gliomas and develop markers to assist in the diagnosis and treatment of glioblastoma.

More and more studies have shown that integrin-associated genes, particularly the ITGB5 gene, can be used to guide patient prognosis and to evaluate therapeutic efficacy. At present, no kit for the ITGB5 gene in glioma is reported. The invention describes the functional action of ITGB5 gene in glioma, and prepares a kit for evaluating adverse prognosis and the effect of radiotherapy and chemotherapy.

Disclosure of Invention

In view of the defects in the prior art, the invention mainly provides an ITGB5 gene-based glioblastoma multiforme aided diagnosis and prognosis evaluation kit and a method for using the same, and finds that there is a significant difference in transcriptional expression levels of the ITGB5 gene in LGG (low-grade glioma) and GBM (glioblastoma multiforme). Compared with LGG tissue, the ITGB5 gene is obviously up-regulated in GBM tissue, and the malignancy degree of glioma patients can be diagnosed by detecting the expression condition of the transcriptional level of the ITGB5 gene. Meanwhile, the prognosis condition and the chemoradiotherapy effect of a patient can be evaluated according to the expression condition of the ITGB5 gene.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows.

An ITGB5 gene-based glioblastoma multiforme auxiliary diagnosis and prognosis evaluation kit comprises a PCR primer pair for amplifying an ITGB5 gene, wherein the primer pair is shown in the specification.

The forward primer sequence is 5'-GGAAGTTCGGAAACAGAGGGT-3'.

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

Preferably, the kit further comprises a PCR primer pair for amplifying the housekeeping gene GAPDH, and the primer pair is.

The forward primer sequence is 5'-GGAGCGAGATCCCTCCAAAAT-3'.

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

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.

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

The kit also comprises a system for reverse transcription of mRNA into cDNA, wherein the reverse transcription reagent 5X RT master mix is PrimeScript RTase, RNase Inhibitor, Random 6 mers, Oligo dT Primer, dNTP mix, reaction Buffer, and a fluorescent quantitative PCR reaction system SYBR Premix Ex TaqTM.

An ITGB5 gene-based glioblastoma multiforme auxiliary diagnosis and prognosis evaluation kit and a using method thereof comprise the following steps.

(1) And (3) treating and grinding the obtained fresh tissue by liquid nitrogen, and then extracting RNA.

(2) The extracted RNA was reverse transcribed into the corresponding cDNA.

(3) The reverse transcribed cDNA was subjected to fluorescent quantitative PCR amplification of ITGB5 and GAPDH genes.

(4) The Ct value of each reaction was recorded using GAPDH as an internal control, and the detection result was expressed as- Δ Ct, where Δ Ct ═ CtGene-CtGAPDH.

Application of ITGB5 gene in preparation of glioblastoma multiforme auxiliary diagnosis and prognosis evaluation kit.

Compared with the prior art, the invention has the following advantages and beneficial effects.

(1) The invention discloses that the ITGB5 gene is related to glioma disease progression for the first time, and the ITGB5 gene is expected to become a molecular marker for diagnosing GBM and provides a new thought for researching the molecular mechanism of glioma disease progression.

(2) The transcriptional expression levels of the ITGB5 genes in LGG and GBM are obviously different, compared with LGG tissues, the ITGB5 gene is obviously up-regulated in GBM tissues, the malignancy degree of a glioma patient can be diagnosed by detecting the transcriptional expression condition of the ITGB5 gene, so that an auxiliary diagnostic kit for glioblastoma containing the ITGB5 gene can be prepared, and the method for diagnosing glioblastoma by detecting the transcriptional expression of the gene is more sensitive and specific, and is favorable for early diagnosis of diseases.

(3) The invention firstly proves that the expression level of the ITGB5 gene in GBM is directly related to the prognosis of a patient, and the survival time of the GBM patient with high ITGB5 is obviously reduced compared with the GBM patient with low ITGB5 expression. By detecting the expression condition of the ITGB5 transcription level, the prognosis condition of a GBM patient can be estimated, so that the glioblastoma multiforme prognosis evaluation kit containing the ITGB5 gene can be prepared, the prognosis of the patient can be more accurately and objectively judged, and the chemoradiotherapy sensitivity of the patient can be evaluated.

(4) The invention also suggests that the glioma disease can be blocked by intervening the expression of ITGB5, and the glioma gene can be used as a specific target point for future targeted therapy of glioma, thereby providing a new idea for the therapy of glioma diseases.

Drawings

FIG. 1 shows that ITGB5 gene expression is correlated with malignant phenotypes such as IDH1 wild type and Mesenchymal (Mesenchymal).

FIG. 2 shows that ITGB5 gene expression can be used as a diagnostic indicator of GBM and mesenchyme (Mesenchymal).

FIG. 3 shows that the levels of mRNA and protein expression of ITGB5 gene are both related to each other in clinical tissue specimens.

FIG. 4 shows that the ITGB5 gene is indicative of a poor prognosis for GBM patients.

FIG. 5 shows ITGB5 participating in the chemoradiotherapy resistance of GBM patients.

FIG. 6 shows that knocking down the expression of ITGB5 can inhibit the migration and invasion capacity of glioma cells.

Detailed Description

The invention is explained in more detail below with reference to specific embodiments and the drawing. 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 are given.

The ITGB5 gene has obvious difference in databases and is a marker of malignant progression of glioma.

The study mainly contained two database platforms, The Chinese Glioma Gene Atlas (CGGA) and The tumor gene Atlas project (The Cancer Genome Atlas, TCGA). CGGA is a database for analyzing samples of glioma patients in China, which is initiated and established by Peking Tiantan hospitals in 2012, is a large-scale database which is peculiar to China and can reveal the molecular mechanism of glioma occurrence and development of Chinese people, and provides a large amount of basis and clinical support for glioma research in China. A total of 172 low-grade glioma (LGG) samples and 138 GBM samples were included in the CGGA database (http:// www. CGGA. org. cn). The ITGB5 gene transcript level results are from Illunima Hiseq 2000 platform RNA-seq results. The TCGA project group was originally composed of The National Cancer Institute (NCI) and The national human genome institute (NHGRI) and developed into a very large data research platform, low-grade glioma (LGG) and Glioblastoma (GBM) as one of The earliest studied tumors with a large number of comprehensive data results. The TCGA RNA sequencing (RNAseq) dataset comprised 625 samples, among which 470 LGG samples and 155 GBM samples.

As shown in FIG. 1A, B, in the databases of CGGA and TCGA RNA-seq, the expression level of ITGB5 gene in GBM patient tissues was significantly increased as compared to that in LGG patient tissues (CGGA RNA-seq: P < 0.0001, TCGA RNA-seq: P < 0.0001); furthermore, ITGB5 was significantly higher in interstitial GBM patients than in classical and proneuronal GBM patients (fig. 1C, D) (×: P < 0.0001); furthermore, studies on the state of IDH1 mutation in GBM patients found that ITGB5 was expressed higher in IDH1 wild-type patients than in mutant patients (fig. 1E, F).

2. Diagnostic ROC curves for GBM and mesenchyme were drawn based on differential expression of the ITGB5 gene in CGGA and TCGA RNA-seq glioma tissue samples.

ROC curves for diagnosing GBM were drawn according to the ITGB5 expression levels of LGG and GBM based on the above CGGA and TCGA RNA-seq glioma tissue samples. 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. AUC has lower accuracy when being 0.5-0.7, AUC has certain accuracy when being 0.7-0.9, and AUC has higher accuracy when being more than 0.9. As shown in FIG. 2A, B, the area under the ROC curve (AUC) of ITGB5 for diagnosing glioblastoma is CGGA RNA-seq: 0.654 (fig. 2A, P < 0.0001), TCGA RNA-seq: 0.647 (fig. 2B, P < 0.0001), can be used as an indicator for the diagnosis of GBM.

In view of the high malignancy and poor prognosis of interstitial GBM, the present study further verifies the diagnosis effect of ITGB5 gene on the interstitial type of GBM patients, and the results are shown in FIG. 2C, D, and the areas under ROC curve (AUC) of the interstitial type of GBM patients diagnosed by ITGB5 are CGGA RNA-seq: 0.878 (FIG. 2C, P < 0.001), TCGA RNA-seq: 0.739 (fig. 2D, P < 0.001), i.e. the diagnosis has a certain accuracy.

3. Clinical tissue samples tested for differential expression of ITGB5 in LGG and GBM.

21 low-grade glioma (LGG) samples and 21 glioblastoma multiforme (GBM) tissue samples, which were pathologically diagnosed at the first hospital affiliated to the university of medical science in China, were collected, and 42 tissue samples were counted. RNA extraction and reverse transcription were performed on these samples, and fluorescent quantitative PCR amplification of ITGB5 and GAPDH genes was performed on the reverse-transcribed cDNA. The results are shown in fig. 3A, where the ITGB5 gene was significantly elevated in GBM patient tissues compared to LGG patient tissues (P < 0.0001). The same tissue samples were further examined by immunohistochemistry (FIG. 3B) and western blot (FIG. 3C) and the results were consistent with the PCR results. The fluorescent quantitative PCR assay in the mature glioma cell line and the primary glioma cell line revealed that the glioma cell line ITGB5 gene expression was significantly increased compared to human astrocytes (NHA) (FIG. 3D), and the western blot results also gave the same results as the fluorescent quantitative PCR (FIG. 3E). The protein level verification shows that the fluorescent quantitative PCR detection of RNA can well reflect the protein expression of ITGB5, and the reliability of the RNA result is verified.

4. GBM diagnostic ROC curves were drawn based on differential expression of ITGB5 in clinical tissues.

ROC curves for diagnosing GBM were drawn based on RNA expression levels of ITGB5 in 42 glioma tissue samples collected above. As a result, as shown in fig. 3F, the area under ROC curve (AUC) of ITGB5 for diagnosing glioblastoma was 0.943, i.e., the diagnosis was highly accurate.

5. Survival curves were plotted for ITGB5 expression levels in the CGGA RNAseq and TCGA RNAseq databases.

In the CGGA RNAseq database, survival curves were plotted for GBM patients in descending order of their mRNA expression levels according to ITGB5, the first 69 cases were low-ITGB 5 expression groups, the last 69 cases were high-ITGB 5 expression groups, and fig. 4A is a survival curve plotted in the CGGA RNAseq database based on the levels of ITGB5 gene mRNA. P is 0.0046 by longrank test analysis, namely the prognosis has statistical significance; in the TCGA RNAseq database, for 155 GBM patients, survival curves were drawn according to the sequence of ITGB5 mRNA expression levels from small to large, the first 77 were low ITGB5 expression groups, the last 78 were high ITGB5 expression groups, and longrank test analysis gave P0.0121, which is statistically significant in the prognosis, as shown in fig. 4B. Further illustrating that in GBM, patients with high expression of ITGB5 have a significantly worse prognosis than patients with low expression of ITGB 5.

6. And (3) aiming at the radiotherapy condition of the patient in CGGA RNAseq and TCGA RNAseq databases, drawing a survival curve according to the ITGB5 expression level.

In the CGGA RNAseq database, GBM patients were ranked from small to large according to their mRNA expression levels of ITGB5, the first 69 cases were low-ITGB 5 expression groups, the last 69 cases were high-ITGB 5 expression groups, and the GBM patients were divided into four groups, i.e., ITGB5 low-expression + radiotherapy, ITGB5 high-expression + radiotherapy, ITGB5 low-expression + non-radiotherapy, and ITGB5 high-expression + non-radiotherapy, according to whether the patients received radiotherapy, and the number of patients per group was 38, 42, 25, and 19, and survival curves were drawn according to these four groups of patients, and fig. 5A is a survival curve of patients who received radiotherapy, drawn based on the ITGB5 gene mRNA level, in the CGGA RNAseq database. Among patients receiving radiotherapy, patients in the low-expression ITGB5 group survived longer than those in the high-expression ITGB5 group, and longrank test analysis P = 0.0009 was statistically significant; in patients who did not receive radiotherapy, the survival time of patients in the low-expression ITGB5 group was not significantly different from that in the high-expression ITGB5 group, and longrank test analysis P = 0.3356 was not statistically significant.

In the TCGA RNAseq database, GBM patients are ranked from small to large according to the mRNA expression level of ITGB5, the first 77 cases are low-ITGB 5 expression groups, the last 78 cases are high-ITGB 5 expression groups, and the GBM patients are divided into four groups, i.e., ITGB5 low-expression + radiotherapy, ITGB5 high-expression + radiotherapy, ITGB5 low-expression + non-radiotherapy, and ITGB5 high-expression + non-radiotherapy, according to whether the patients receive radiotherapy, wherein the number of people in each group is 64, 63, 9, and 12, and survival curves are drawn according to the four groups of patients, and fig. 5B is a survival curve of patients who receive radiotherapy in the TCGA RNAseq database based on the ITGB5 gene mRNA level. Among patients receiving radiotherapy, the survival time of patients in the low-expression ITGB5 group is higher than that of patients in the high-expression ITGB5 group, and the longrank test analysis P = 0.0171 has statistical significance; in patients who did not receive radiotherapy, the survival time of patients in the low-expression ITGB5 group was not significantly different from that in the high-expression ITGB5 group, and longrank test analysis P = 0.4501, which has no statistical significance. The ITGB5 expression high-low survival curves drawn by CGGA RNAseq and TCGA RNAseq databases aiming at the radiotherapy condition of the patient show that the ITGB5 participates in the glioma radiotherapy resistant process, and the radiotherapy sensitivity of the GBM patient can be predicted according to the ITGB5 expression quantity.

7. For the chemotherapy condition of the patients in the CGGA RNAseq and TCGA RNAseq databases, a survival curve is drawn according to the expression level of ITGB 5.

In the CGGA RNAseq database, GBM patients were ranked from small to large according to their mRNA expression levels of ITGB5, the first 69 cases were low-ITGB 5 expression groups, the last 69 cases were high-ITGB 5 expression groups, and the patients were divided into four groups of ITGB5 low-expression + chemotherapy, ITGB5 high-expression + chemotherapy, ITGB5 low-expression + non-chemotherapy, and ITGB5 high-expression + non-chemotherapy according to whether they received chemotherapy, and the number of patients in each group was 45, 38, 18, and 23, and survival curves were drawn according to these four groups of patients, and fig. 5C is a survival curve of patients who received chemotherapy in the CGGA RNAseq database based on the level of ITGB5 gene mRNA. Among patients receiving chemotherapy, patients with low-expression ITGB5 survived longer than those with high-expression ITGB5, and longrank test analysis P = 0.0058 was statistically significant; in patients who did not receive chemotherapy, the survival time of patients in the low-expression ITGB5 group was not significantly different from that in the high-expression ITGB5 group, and the longrank test analysis P = 0.8661 had no statistical significance. In the TCGA RNAseq database, GBM patients are ranked from small to large according to the mRNA expression level of ITGB5, the first 77 cases are low-ITGB 5 expression groups, the last 78 cases are high-ITGB 5 expression groups, and the GBM patients are divided into four groups, i.e., ITGB5 low-expression + chemotherapy, ITGB5 high-expression + chemotherapy, ITGB5 low-expression + non-chemotherapy, and ITGB5 high-expression + non-chemotherapy, according to whether the patients receive chemotherapy, wherein the number of the patients in each group is 56, 55, 11, and 18, and survival curves are drawn according to the four groups of patients, and fig. 5D is a survival curve of the patients who receive chemotherapy in the TCGA RNAseq database based on the ITGB5 gene mRNA level. Among patients receiving chemotherapy, patients with low-expression ITGB5 group survived longer than those with high-expression ITGB5 group, and longrank test analyzed P = 0.0072, which is statistically significant; in patients who did not receive chemotherapy, the survival time of patients in the low-expression ITGB5 group was not significantly different from that in the high-expression ITGB5 group, and the longrank test analysis P = 0.5297 had no statistical significance. The ITGB5 expression high-low survival curves drawn by CGGA RNAseq and TCGA RNAseq databases aiming at the chemotherapy condition of patients indicate that the ITGB5 participates in the glioma chemotherapy resistance process, and the chemotherapy sensitivity of GBM patients can be predicted according to the ITGB5 expression quantity.

8. Cox multifactorial regression analysis based on CGGA RNAseq and TCGA RNAseq database GBM.

Age, IDH1 mutation status, whether to receive radiation therapy, whether to receive chemotherapy, and the expression level of ITGB5 were included in a Cox's multi-factor regression analysis model and these factors were analyzed for relationship to GBM prognosis. The results are given in table 1 (multifactor COX analysis suggests that the model can be used as CGGA RNAseq, an independent prognostic factor in GBM database), table 2 (multifactor COX analysis suggests that the model can be used as CGGA RNAseq, an independent prognostic factor in GBM database).

Table 1. and table 2 the results of the analyses are: the results obtained in the CGGA RNAseq and TCGA RNAseq databases indicate that ITGB5 expression levels are of independent prognostic value for GBM (CGGA: P ═ 0.0039, TCGA: P ═ 0.0132).

9. The effect of ITGB5 on glioma invasion and migration was examined in glioma cell lines and glioma primary cell lines.

The ITGB5 small interfering RNA is constructed, the knockdown effect of two pairs of siRNA is verified in a glioma cell line LN229 and a glioma primary cell line DGC1228 respectively, as shown in FIG. 6A, the mRNA level of ITGB5 in LN229 and DGC1228 cell lines is remarkably down-regulated by two pairs of small interfering RNA of siITGB5-368 and siITGB5-1218 compared with a control group, and FIG. 6B further proves that the expression of ITGB5 in LN229 and DGC1228 cell lines is remarkably down-regulated by two pairs of small interfering RNA of siITGB5-368 and siITGB5-1218 compared with the control group through a western blot method. As shown in fig. 6C, as a result of Transwell experiments, it was found that siITGB5-368 and siITGB5-1218 compared with a control group, down-regulating ITGB5 expression can significantly inhibit migration and invasive ability of LN229 and DGC1228, the invasive and migratory ability directly reflects the recurrence of GBM patients, ITGB5 expression is correlated with glioma invasive migration ability, and the recurrence time of GBM patients can be predicted by ITGB5 expression amount.

10. And (3) preparation of an ITGB5 gene-based glioblastoma multiforme auxiliary diagnostic kit.

The glioblastoma multiforme auxiliary diagnosis kit comprises a PCR primer pair for amplifying an ITGB5 gene, a PCR primer pair for amplifying a housekeeping gene GAPDH, a SYBR Green polymerase chain reaction system, an RNA extraction reagent and a system for reverse transcription of mRNA into cDNA.

The PCR primer pair for amplifying the ITGB5 gene is shown in the specification.

An ITGB5 gene-based glioblastoma multiforme auxiliary diagnosis and prognosis evaluation kit comprises a PCR primer pair for amplifying an ITGB5 gene, wherein the primer pair is shown in the specification.

The forward primer sequence is 5'-GGAAGTTCGGAAACAGAGGGT-3'.

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

The kit also comprises a PCR primer pair for amplifying housekeeping gene GAPDH, wherein the primer pair is shown in the specification.

The forward primer sequence is 5'-GGAGCGAGATCCCTCCAAAAT-3'.

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

The SYBR Green polymerase chain reaction system comprises 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.

An application method of the glioblastoma multiforme auxiliary diagnosis kit based on the ITGB5 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 at 4 ℃ for 15min, and the upper aqueous phase was transferred to a new EP tube (ca. 500 ul).

3) Adding isopropanol (about 500ul, precooled at 4 deg.C) with the same volume as the supernatant, mixing by inversion, standing at room temperature for 10-20min (average 15min), 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 5 min.

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) And (4) a reverse transcription system.

1ug RNA volume: x ul.

5X PrimeScript RT Master Mix: 4ul。

RNase free water: 16-Xul.

The total system is as follows: 20 ul.

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

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 is provided.

SYBR Premix Ex TaqTM: 10ul。

The upstream primer (10 umol) is 1 ul.

The downstream primer (10 umol) is 1 ul.

cDNA template: 2 ul.

RNase free water: 6 ul.

The total system is as follows: 20 ul.

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) Setting a program: the total of 40 cycles of 95 ℃ 10min95 ℃ 10min, 95 ℃ 5S, 60 ℃ 10S, 72 ℃ 10S + dissolution profile.

11) And (3) analyzing experimental data: with GAPDH as an internal reference, the Ct value for each reaction was recorded as the number of cycles that the fluorescence signal in each reaction tube went through to reach the set threshold. The expression was higher as the Δ Ct is smaller as the initial copy number is higher and the expression is higher, and the expression level of ITGB5 was used as an index.

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.

Claims (6)

1. An application of an ITGB5 gene in preparation of a glioblastoma multiforme auxiliary diagnosis and prognosis evaluation kit is characterized by comprising a PCR primer pair for amplifying an ITGB5 gene, wherein the primer pair is as follows:

the forward primer sequence is 5'-GGAAGTTCGGAAACAGAGGGT-3'

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

2. The use of claim 1, wherein the kit further comprises a PCR primer pair for amplifying the housekeeping gene GAPDH, wherein the primer pair is:

the forward primer sequence is 5'-GGAGCGAGATCCCTCCAAAAT-3'

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

3. The use of claim 1, wherein the kit comprises a SYBR Green polymerase chain reaction system comprising PCR buffers, dNTPs, SYBR Green fluorescent dyes, enzyme-free water and a fluorescent quantitation plate.

4. The use of claim 1, wherein the kit comprises an RNA extraction reagent comprising Trizol, chloroform, isopropanol, 75% ethanol and RNase free water.

5. The use of claim 1 wherein the kit comprises a system for reverse transcription of mRNA into cDNA and the reverse transcription reagent 5X RT master mix is 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 according to claim 1, wherein the method of use of the kit comprises in particular the steps of:

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

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

(3) performing fluorescent quantitative PCR amplification on ITGB5 and GAPDH genes by using the reverse transcribed cDNA;

(4) the Ct value of each reaction was recorded using GAPDH as an internal control, and the detection result was expressed as- Δ Ct, where Δ Ct ═ CtGene-CtGAPDH.

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