| Backgroud:Glioma is the most common malignant tumor in the central nervous system,which derives from the glial cells.Glioma is characterized by rapid progression,resistance to chemotherapy and easy replace after surgery.Recently,with the development in molecular biology,the pathogenesis and treatment of glioma have been further investigated.The discovery of many factors,including IDH,1p/19q codeletion,H3 Lys27Met,and RELA-fusion,will help in the diagnosis,grading and treatment of brain tumors.Nowdays,the research of glioma mainly focuses on molecular-targeted therapy,immunotherapy,gene therapy and novel drug delivery technology.Cyclin-dependent Kinases(CDKs),the key kinases in cell cycle period,play the key role in rapid proliferating tumor cells.Overexpression of cell cycle-associated CDKs leads to hyperphosphorylation a variety of substrates of tumor cells that support the proliferation of the cells in an poor extracellular or intracellular environment.In some cases,CDKs inhibitor proteins are inactivated by point mutation deletion or silencing,resulting in increased activity of CDKs,transformation the cell cycle of normal cells leading to malignant clone.The present study aims to clarify the clinical significance of CDK4,CDK6,CDK7 in the diagnosis,treatment and grading of glioma,and conduct an experiment on glioma U251 cells using CDK7 inhibitor THZ1 combined with radiotherapy in vitro.It can provide theoretical basis for understanding the clinical treatment of glioma.Objective:The expression of CDK4,CDK6,CDK7 in glioma of different grades was studied by CGGA database(http://www.cgga.org.cn/)The expression of CDK4,CDK6,CDK7 in clinical glioma tissues and paracancerous tissues was investigated by immunohistochemistry.The relevance between CDK4,CDK6,CDK7 and WHO classification was studied in patients with glioma.The correlation between CDK4,CDK6,CDK7 and objective indicators such as age and sex was also investigated.In vitro the effect of CDK7 inhibitor THZ1 on glioma U251 cells was studied and the synergistic effect of THZ1 combined with radiotherapy was analvzed in U251 cells.Method:Clinical data including CDK4,CDK6,CDK7 expression and WHO grade were downloaded from CGGA database.The KrusKal-Wallis test was used to analyze the statistical difference between the expression level and WHO grade of glioma.Patients with glioma admitted to the Department of Oncology and Neurosurgery of the Second Hospital of Shandong University from January 2014 to December 2019 were selected.All patients underwent surgical resection,and the diagnosis of glioma was confirmed by pathologists after surgery.A total of 67 patients were enrolled in this study.The data were obtained by immunohistochemistry.Glioma tissues group and paracancerous tissues group were confirmed by pathologic.The differences of CDK4,CDK6,CDK7 expression between the glioma tissues and the paracancerous tissues were analyzed by chi-square test.The relevance between CDK4,CDK6,CDK7 expression and age,sex,grade in glioma tissues were analyzed by chi-square test.The survival data from CGGA database were used to analyze the overall survival(OS)difference between high expression group and the low expression group by univariate Kaplan-Meier survival analysis.Two-factor variance analysis of the inhibitory effect of THZ1 in different concentrations on glioma U251 cells at different time points.Two-factor variance analysis of the effect of THZ1 combined with radiotherapy on U251 cells with the experiment of clone formation.Chi-square test of R×C tandem table was used to analyze the difference of cell cycle changes under the condition of THZ1 and radiotherapy.The expression differences of CDK7,Bax and y H2AX proteins were analyzed by independent sample t test following THZ1 and radiotherapy treatment.Results:According to CGGA database analysis,the differences between WHO Ⅱand WHO Ⅲ,WHO Ⅱ and WHO Ⅳ of CDK4 and CDK7 level were statistically significant(P<0.05).The expression of CDK6 gene was statistically significant in patients with different WHO grades(P<0.05).Immunohistochemistry reselts showed that the expression of CDK4,CDK6,CDK7 was significantly different between glioma tissues and paracancerous tissues(P<0.05).Clinical data show that the expression of CDK4 in glioma tissues was not relevant to WHO grade.CDK6,CDK7 expression was correlated with WHO grade of glioma,and the difference was statistically significant(P<0.05).However there was significant with age and sex(P>0.05).CDK4,CDK6,CDK7 expression was correlated with overall survival(OS),and the difference was statistically significant(P<0.001).MTT assay results showed that THZ1 could inhibit the prolifertation of glioma U251 cells.The inhibitory effect was enhanced with the increase drug concentration.Cloning and formation experiments showed that THZ1 could enhance the effect of X-ray on U251 cells,and the difference between the irradiation group and the radiation group was statistically significant(P<0.001).The radiosensitization ratio(SER)was 1.478.Western blot showed that THZ1 could inhibit the expression of CDK7 in glioma U251 cells.THZ1 combined with X ray could increase the expression of Bax yH2AX,and the difference was statistically significant(P<0.05).Cell cycle assay showed that THZ1 combined with X-ray could arrest the cell cycle of glioma U251 from G0/G1 phase to G2/M phase(P<0.001).Conclusion:CDK4,CDK6,CDK7 protein was highly expressed in glioma tissues compared to pericarcinomatous tissues.The expression of CDK6,CDK7 protein,was higher in the high grade glioma than in the low grade glioma.The expression level of CDK4,CDK6,CDK7 protein is related to the overall survival of patients.The high expression group,possess shorter survival time.THZ1 can inhibit proliferation and induce apoptosis of glioma U251 cells.The combination of THZ1 and X-ray can significantly inhibit the proliferation of U251 cells,which may be induce to the cell cycle change induced by the inhibition of CDK7 protein expression by THZ1.CDK7 may be a key factor in the diagnosis,treatment and prognosis of glioma patients. |
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