| Background and objective Glioma is the most common primary malignant tumor in the central nervous system,it has high recurrence rate and mortality.Despite recent advances in surgery,adjuvant radiochemotherapies for glioma,the prognosis for patients with high-grade glioma is still grim,the median survival of patients with glioblastoma is about 14.6 months after standard treatment,and 5 years survival rate is less than 10%.In order to improve the therapeutic effect,more and more researches have focused on the molecular regulation mechanism of glioma development and the new effective therapeutic targets.Mammalian Sterile 20 Like Kinase 1(MST1)is one of the core components of Hippo signaling pathway in tumor suppressor signaling pathway,which is involved in apoptosis.It plays an important role in regulating the progress of cell cycle and inhibiting tumorigenesis.Many studies show that MST1 was abnormal down-regulated in many human tumors,and was associated with tumorous clinical stages,pathologic grade and prognosis of patients.However,the expression of MST1 in gliomas was not similar to that in other tumors.The expression of MST1 protein was heterogeneous in gliomas,with big differences.MST1 level of each glioma sample was high and low compared with the normal brain tissues.Therefore,the expression of MST1 and its relationship with clinical and pathological parameters in gliomas remains unclear.Studies have shown that DNA methylation mediated by DNA methyltransferase1(DNMT1)play an important role in the silence of many key tumor suppressor genes;transforming growth factor-β(TGF-β)can promote the growth of glioma cells and is related to the malignant degree of glioma,and TGF-β can also regulate expression of DNMT1 in lung fibrosis,prostate cancer,and lung cancer.Therefore,we hypothesized that TGF-β increases DNMT1 expression,and then DNMT1-mediated MST1 methylation induces down-regulation of MST1 expression and cell proliferation and invasion in glioma.Therefore,in this study,the expression of MST1 in glioma specimens were detected by immunohistochemistry,and the correlation between the expression of MST1 and tumourous clinical pathological parameters and prognosis of patients were analyzed;Then,effects of TGF-β and DNMT1 on MST1 expression and the corresponding cell proliferation and invasion were examined in glioma cells,and the relationship among TGF-β,DNMT1 and MST1 were explored.The aim of this study is to verify the above hypothesis through the following experiments to determine the expression of MST1 in gliomas and explore the molecular mechanism of its regulation,and to provide theoretical basis for molecular mechanism and molecular targeted therapy in the development of glioma.Methods 1.A total of 120 gliomas and 5 normal brain paraffin specimens from 2009 to 2016 in the Second Affiliated Hospital of Anhui Medical University were selected and the corresponding clinical and follow-up data were collected.The expression of MST1 in 120 cases of gliomas and 5 normal brain tissues were detected by immunohistochemical two step method,and the correlation between the expression of MST1 and clinicopathological parameters and prognosis of patients was analyzed by chi-square test.The cumulative probability of survival was calculated using the Kaplan-Meier method,and the log-rank test was used to compare survival curves.2.U87 and U251 glioma cells were treated with exogenous TGF-β,5aza-2-deoxycytidine(5-Azad C),and the m RNA and protein expression of MST1 and DNMT1 were measured by real time quantitative PCR and western blot at 24 h and 48 h after treatment,then U251 and U87 glioma cells were divided into three groups according to the drug treatment manners: 1)the control group(control): do not add drugs,instead of PBS;2)TGF-β groups: add 5 ng/m L TGF-β;3)TGF-β + 5-Azad group C: add 5 ng/m L TGF-β,and then add 5 umol/L 5-Azad C after 24 h.The MST1 m RNA and protein expression of U251 and U87 cells were separately measured by real time quantitative PCR and western blot in three groups,and proliferation,migration and invasion of U251 and U87 cells were separately measured by CCK8 and Transwell assay in three groups.The lentiviral vector system expressing DNMT1 sh RNA or negative control sh RNA were constructed,and then U87 and U251 cells were infected with the lentivirus constructs,and the DNMT1 and MST1 expression were measured by real time quantitative PCR and western blot after transfection to determine the efficiency of DNMT1 gene silencing and the expression of MST1 after silence of DNMT1,then U251 and U87 glioma cells were divided into three groups: the control groups(sh Con),TGF-β+sh Con groups,sh DNMT1+TGF-β groups,The m RNA and protein expression of MST1 of U251 and U87 cells were separately measured by real time quantitative PCR and western blot in three groups,and proliferation,migration and invasion of U251 and U87 cells were separately measured by CCK8 and Transwell assay in three groups.Results 1.Expression of MST1 in human normal brain and glioma tissues.Positive expression of MST1 was detected in 54 cases,and negative expression in 66 cases in 120 cases of glioma tissues,positive expression rate was 45.0%;the positive expression of MST1 in 5 cases of normal brain tissues was 5,and the positive expression rate was 100%.Positive signal was mainly detected in the cytoplasm.2.The relationship between the expression of MST1 and the clinical and pathological parameters of glioma patients.Positive expression of MST1 in high grade gliomas(grade III-IV)and low grade gliomas(grade I-II)were 21 and 33 cases,the positive expression rate were 33.3% and 57.9%;The positive expression rate in high grade gliomas was significantly lower than that in low grade gliomas(P=0.007).The expression of MST1 was not correlated with age,sex,preoperative KPS(Karnofsky performance score)and tumor size of the patients.3.The relationship between the expression of MST1 and the prognosis of glioma patients.Kaplan-Meier and log-rank analysis showed that the median survival time of patients with positive and negative expression of MST1 were 48 months and 20 months respectively in all patients.Patients with negative expression of MST1 had poor overall survival,compared with those with positive expression of MST1.The difference was statistically significant(P=0.000).Kaplan-Meier and log-rank analysis showed that the median survival time of patients with positive and negative expression of MST1 were 18 months and 11 months respectively in 63 cases of high grade gliomas.Patients with negative expression of MST1 had poor overall survival,compared with those with positive expression of MST1.The difference was statistically significant(P=0.010).4.TGF-β can reduce MST1 expression and increase DNMT1 expression.The expression of MST1 m RNA and protein were significantly reduced at 24 h and 48 h after TGF-β treatment in glioma cells(*P<0.05,**P<0.01).On the contrary,the expression of DNMT1 m RNA were significantly increased(**P<0.01).Moreover,the expression of MST1 decreased gradually with the prolongation of TGF-β duration of action,the expression of DNMT1 increased gradually with the prolongation of TGF-β duration of action.TGF-β induced down-regulation of MST1 expression and up-regulation of DNMT1 expression both in a time-dependent manner.5.5-Azad C can increase MST1 expression and reverse reduction of MST1 expression induced by TGF-β and inhibit the proliferation,migration and invasion of glioma cells mediated by TGF-β The expression of MST1 m RNA and protein were significantly increased at 48 h after 5-Azad C treatment in glioma cells(**P<0.01),MST1 m RNA and protein expression of U251 and U87 cells were significantly decreased in TGF-β groups compared with the control groups(**P<0.01),MST1 m RNA and protein expression of U251 and U87 cells were significantly increased in the TGF-β+5-Azad C groups compared with the TGF-β groups(##P<0.01);proliferation,migration and invasion of U251 and U87 cells were significantly increased in the TGF-β groups compared with the control groups(**P<0.01),proliferation,migration and invasion of U251 and U87 cells were significantly decreased in the TGF-β+5-Azad C groups compared with the TGF-β groups(##P<0.01).6.DNMT1 silence mediated by lentiviral vector can increase MST1 expression and reverse reduction of MST1 expression induced by TGF-β and inhibit TGF-β-mediated the proliferation,migration and invasion of glioma cells.We successfully constructed the lentiviral vector system expressing DNMT1 sh RNA or negative control sh RNA,and then U87 and U251 cells were infected with the lentivirus constructs,U87 and U251 glioma cells infected with Lv-sh DNMT1 exhibited significantly inhibited the expression of DNMT1 compared with Lv-sh Con-infected cells(**P<0.01),DNMT1 gene silence efficiency is high.On the contrary,the expression of MST1 of Lv-sh DNMT1-infected cells was significantly increased compared with Lv-sh Con-infected cells(**P<0.01);MST1 m RNA and protein expression of U251 and U87 cells were significantly decreased in TGF-β+sh Con compared with the sh Con groups(**P<0.01),MST1 m RNA and protein expression of U251 and U87 cells were significantly increased in the sh DNMT1+TGF-β groups compared with the TGF-β+sh Con groups(#p<0.05,##P<0.01);proliferation,migration and invasion of U251 and U87 cells were significantly increased in the TGF-β+sh Con groups compared with the sh Con groups(**P<0.01),proliferation,migration and invasion of U251 and U87 cells were significantly decreased in the sh DNMT1+TGF-β groups compared with the TGF-β+sh Con groups(#p<0.05,##P<0.01).Conclusion 1.The expression of MST1 was not related with age,sex,preoperative KPS score and tumor size of the patients.2.The expression of MST1 was correlated with the pathological grade of tumor,the positive expression rate of MST1 in high grade gliomas was significantly lower than that in low grade gliomas.3.The overall survival rate of negative expression of MST1 was significantly lower than that of positive expression of MST1.4.TGF-β increases DNMT1 expression,and then epigenetic repression of MST1 mediated by DNMT1 induces down-regulation of MST1 expression;TGF-β-mediated repression of MST1 by DNMT1 promotes proliferation and invasion in glioma. |
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