The Research Of The Mechanism Of EZH2 Regulating The Chemosensitivity Of Gbm Cells To TMZ

Brain glioma is the most common primary intracranial tumors and about 2/3 of them are malignant glioma. Despite comprehensive measures of conventional surgery, radiotherapy and chemotherapy have made considerable progress, the 2-year survival rate of glioblastoma multiforme(GBM) is still less than 30%. Clinically, the presence phenomena of significantly different prognosis of glioma patients often appear at the same pathological level and the same treatment, the reason may be related to intrinsic biological properties of the tumor as well as its sensitivity to chemotherapy. Recently glioma chemotherapy gradually get attention, TMZ has become adjuvant chemotherapy in first-line drugs, and therefore the factor of affecting chemosensitivity and to exploring the mechanism have also become one of the greatly concerned topics of Clinical Neurosurgery。Histone methyltransferase EZH2,as the catalytic subunit of PRC2, can add three methyl groups on the 27’lysine of histone 3. This 3 methylated histones can result in chromatin condensation and many of oncogene or tumor suppressor gene,which is related to the tumor proliferation, invasion and angiogenesis, activated or silence in the epigenetic.In this reaserch,we plan to further explore the role of EZH2 in the chemosensitivity of glioblastoma to temozolomide.In the vitro study,differential expression of EZH2 of three glioblastoma cell lines was analyzed, GBM that transfected with the constructed si-EZH2 Lentiviral establish different EZH2 expression of GBM cell lines, and the sensitivity of temozolomide of them is detceted, while to further detect the relationship between the expression level of EZH2 and DNA repair protein.And by inhibiting the critical proteins in DNA damage repair pathway to detect the change of chemosensitivity of GBM to temozolomide in order to investigate the role of DNA damage repair pathways in the EZH2 regulating chemoresistance in GBM. All experiments include two parts:Part I is the relationship between EZH2 and glioma cells to TMZ chemosensitivity and Part II is The role of DNA repair pathway in EZH2 regulating the chemosensitivity of GBM cells to TMZ.PART I The relationship between EZH2 and the chemosensitivityof GBM cells to TMZObjectiv:To explore relations between EZH2 and chemosensitivity of glioma, and verify the change of chemosensitivity of the EZH2 knockdown GBM cell line to TMZ.Methods:Real-time PCR and Western-blot method were applied to detect m RNA and protein expression levels of EZH2 in three glioblastoma cell lines and CCK-8 was applicated to detect the different IC50 values of TMZ in the three GBM cell lines. Construct EZH2 RNA interference lentivirus and transfected them into GBM cell lines,then detect the transfection efficiency by flow cytometry and the knockdown efficiency is detected by RT-PCR and Western-blot.At last the sensitivity of EZH2 knockdown cell lines to TMZ is detected by CCK-8 method.Results:The order of EZH2 m RNA expression levels is U251> T98> U87.They did not reach statistical significance(P> 0.05) between the U251 and T98, but did reach statistical significance(P <0.05) between U87 and U251 as well as between U87 and T98. EZH2 protein levels consistent with the m RNA expression levels of EZH2 trend. The TMZ IC50 values of U251, T98 and U87 are(45.5±1.3)ug/ml、(41±2.1)ug/ml and(34.8±5)ug/ml. They did not reach statistical significance(P> 0.05) between U251 and T98, but did reach statistical significance(P <0.05) between U87 and U251,as well as between U87 and T98. The transfected rates of Lentiviral transfected cells U87, U251 cells measured by flow cytometry reached more than 99%, and its corresponding knockdown efficiency of EZH2 protein are 95% and 50%. In U87 and U251, IC50 values of EZH2 knockdown cell lines compared with blank group did reach statistical significance(P <0.05).Conclusion: The expression levels of EZH2 and its sensitivity to temozolomide are negatively correlated.EZH2-knockdown can increase the sensitivity of glioma cells to TMZ.PART II The role of DNA repair pathway in EZH2 regulating the chemosensitivityof GBM cells to TMZ.Objective: To detect the DNA repair levels of EZH2 gene knockdown cell lines and its correlation with the sensitivity change of glioma cells to TMZ.Methods: In EZH2 knockdown cell line of U87 and U251, the protein level of MGMT,MSH2, MSH6, ERCC1, XRCC1 and PARP1 was detected by Western-blot, and the IC50 values change of(si-EZH2+PARP1 i U87) and(si-EZH2+PARP1 i U251)are measured by CCK-8 method.Results: The XRCC1,MSH2, MSH6 and PARP1 protein levels of EZH2 knockdown U87 cell lines were significantly lower than blank group(P <0.05). The MGMT and PARP1 protein levels of EZH2 knockdown U251 cell lines were significantly lower than blank group(P <0.05).In U87 and U251 cell lines,the IC50 values of(si-EZH2 + PARP1 i)group were significantly lower(P <0.05) than those of si-EZH2 group or PARP1 i group which compared with the blank group were significantly lower(P <0.05).After TMZ treatment PARP1 protein levels of U87,U251 of blank group increased more significantly than si-EZH2 group(P<0.05) with dosing time prolonging。Results show that after inhibiting DNA repair protein PARP1,the chemosensitivity of U87 and U251 cells to TMZ has increased significantly;The chemosensitivity of(si-EZH2 + PARP1 i)groups in U87 and U251 to TMZ has increased most obviously. With time of TMZ induction prolonging,PARP1 protein of EZH2 knockdown group, compared with EZH2 unknockdown group,has increased significantly.Conclusion : The expression of EZH2 protein can affect the expression levels of DNA repair protein, suggesting that EZH2 gene may be involved in the DNA repair process. PARP1 expression regulated by the experssion level of EZH2 may be one of the molecular mechanisms of EZH2 regulating the chemosensitivity of GBM cells to TMZ.