The Mechanism Of STMN1Regulating The Chemosensitivity Of Glioma Cells To TMZ

Glioma is the most common malignant tumors of the central nervous system, accountingfor about45to55%of primary intracranial tumors. With the continuously development ofdiagnosis and treatment techniques，Including brain imaging, surgery, radiotherapy andchemotherapy, the prognosis of glioma patients has not been significantly improved, and theaverage survival time for GBMs patients is less than1year. Glioma pathological level, theextent of surgical resection and radiotherapy and chemotherapy sensitivity are the mainfactors affecting the prognosis of patients with glioma. In fact, we can often see some patientswith the same pathological level and treatment options, including total surgical resectionand identical radiotherapy and chemotherapy programs, but there are completely differentprognosis, some Some people quickly recurring，others obtaining long-term survival.It may berelated to differences of individual sensitivity to radiotherapy and chemotherapy. Themolecule mechanisms of glioma chemosensitivity are extremely complex, involving multiplegenes, multiple factors, multiple molecular events. So it is necessary to explore the molecularmechanisms of glioma chemosensitivity, new research ideas and theoretical basis to improveglioma patients chemotherapy, in order to search new research ideas and theory and improveglioma chemotherapy efficacy.STMN1(Stathmin1) was firstly reported by Sobel A in1983, and was studied more inrecent years. STMN1is a kind of microtubule depolymerization phosphoprotein, involving inmany signaling pathways. It can regulate microtubule dynamics balance through itsphosphorylation. STMN1are upregulating in many tumor tissues, so it is also known asoncoprotein18(OP18). In normal cells, STMN1is closely related to the cell cycle, apoptosisand cell motility etc. And in some malignant tumors, high STMN1expression be involved inthe malignancy, proliferation, invasion and metastasis of the tumors, and the patients’ poorresponse to chemotherapy drug and prognosis. Previously, we found that STMN1was one ofthe glioma chemotherapy-sensitive gene though microarray technology combined withbiological analysis. STMN1is not only concerned with glioma biological characteristics such as proliferation, apoptosis, invasion and the cell cycle of glioma, but it can also change gliomacells’ sensitivity to chemotherapeutic drugs significantly if it is knocked down in vitro, andthe mechanisms are not clear.The objective of this research is to study the role of autophagy and apoptosis in theprocess of STMN1regulating the glioma chemosensitivity. In this study, We screen STMN1expression differences between four GBMs cell lines. Then, we build STMN1RNAilentivirus and over-expression vector and transfect them into GBMs cell lines in order toestablish STMN1different expression levels glioma cell lines. The sensitivity of all these celllines to TMZ are measured by CCK-8. Further, autophagy and apoptosis levels are detectedand observed in the STMN1-knockdown glioma cell lines. And finally, The sensitivity of theSTMN1-knockdown glioma cell lines to TMZ are detected by CCK-8after the inhibition ofautophagy and apoptosis. The experiment was divided into three parts as follows.Part I The relationship between STMN1expression level of glioma cells and itschemosensitivity to TMZObjective: To investigate the relationship between STMN1expression level of gliomacells and its chemosensitivity to TMZ，including STMN1-knockdown and over-expressionglioma cell lines. that is, to study the role of STMN1in glioma chemosensitivity to TMZ.Methods: utilizing Real-time quantitative PCR and Western-blot technology detectSTMN1mRNA and protein expression levels in four GBMs cell lines. The cell viability ofthe four mentioned cell lines to TMZ is determined by CCK-8method. Then, build STMN1RNAi lentivirus and over-expression vector and transfect them into tumor cells to establishSTMN1different expression levels glioma cell lines. The transfection efficiency is detectedby Real-time quantitative PCR and Western-blot. Finally, its sensitivity to TMZ determinedby CCK-8method.Results: STMN1mRNA expression levels were U251-MG> A172> U87-MG> T98G, andthey did not reach statistically significant difference (P>0.05) between any two. The STMN1 protein expression differences are consistent with STMN1mRNA expression level trend.T98G was the lowest, apart from the A172group，there is a significant statistical difference(P<0.05) comparing with the other two groups, there was highly significant statisticaldifference between T98G and U251-MG groups (P<0.01). There were significant differences(P<0.05) between the A172and U87-MG groups. U251-MG, A172, U87-MG and T98Gwhich are treated with TMZ for72hours, the cell viability were decreased, but it does notreach statistical difference (P>0.05) between any two. The interference efficiency ofU251-MG and T98G are both approximately60%,and the overexpression levels of STMN1of the two cell lines groups are about2times more than corresponding blank control groups.The U251+STMN1-knockdown+TMZ group treated with TMZ for72hours wassignificantly lower than the U251blank group (P<0.01).T98+STMN1-overexpression+TMZ group shows a downward trend with incubation timeincreasing, and the cell viability change does not reach statistically significance (P>0.05).Conclusion: To some extent, STMN1expression levels of glioma cells and its sensitivityto TMZ are negatively correlated. In U251-MG, STMN1-knockdown can increase thechemosensitivity to TMZ. In T98G cell line, there is no certain STMN1expression level andits chemosensitivity to TMZ. Part II The relationship between apoptosis and STMN1regulating the chemosensitivityof glioma cells to TMZObjective: To investigate the role of apoptosis in the process of STMN1regulating gliomachemosensitivity. In other words，whether apoptosis involve in the network of the increasedsensitivity to TMZ of U251-MG when STMN1are knockouted targetly.Methods: Select STMN1-knockdown U251-MG cell line as the subject, and using flowcytometry detect the apoptosis rate after delivering TMZ and cisplatin72hours. Inhibiting apoptosis with Z-VAD (OMe)-FMK, then applying CCK-8assay determine the cell’ssensitivity to TMZ.Results:The apoptosis rate of U251-MG(RNAi STMN1)+TMZ group is high than that ofU251-MG(RNAi STMN1) cell line, which reaches the statistical significant difference (P<0.05), but it lower than that of U251-MG(RNAi STMN1)+Cisplatin group, reachingstatistical highly significant difference (P <0.01). The OD value of (Z-VAD (OMe)-FMK+TMZ) group was significantly lower than in the control group (P <0.05). The cell viability ofU251(RNAi-STMN1)+(Z-VAD (OMe)-FMK+TMZ) group was significantly higher thanthe U251(RNAi-STMN1)+TMZ group (P <0.05). The OD value of U251(RNAi-STMN1)+TMZ group was highly significantly lower than U251(RNAi-STMN1) group (P <0.01).Conclusion: TMZ can induce apoptosis in GBM cell, but apoptosis rate was significantlylower than this induced by cisplatin. Inhibiting apoptosis, the sensitivity to TMZ of U251(RNAi-STMN1) decreases, indicating that apoptosis may involved in this process thatSTMN1regulating glioma chemosensitivity. Part Ⅲ The relationship between apoptosis and STMN1regulating thechemosensitivity of glioma cells to TMZObjective: To investigate the role of autophagy in the process of STMN1regulating gliomachemosensitivity to TMZ.Methods: using TEM observe autophagy ultrastructure—autophagolysosome after TMZinduction in U251-MG (RNAi-STMN1). Western-blot was used to detect U251-MG andA172TMZ-induced autophagy-related gene/protein LC3, Beclin1and ATG5expressionlevels. to observe the TMZ-induced autophagic vacuoles in U251-MG through GFP-LC3Btracer method under laser scanning confocal microscope (RNAi-STMN1), as well asfluorescence intensity changes after the suppression of autophagy by3-MA or bafilomycin A1. And Quantitative Real-time PCR and Western-blot method are used to detectautophagy-related gene and protein expression levels. Finally, the sensitivity to TMZ ofU251-MG (RNAi-STMN1) is assayed by CCK-8method after the inhibition of autophagy by3-MA and/or bafilomycin A1early and late.Results: After TMZ induction, autophagic vacuoles emerge in cytoplasm of U251-MG. ForU251-MG (Si-STMN1) after TMZ induction, The degree of cell damage is worse thanU251-MG and U251-MG(Scramble), and autophagic vacuoles in the cytoplasm increase. InU251-MG and A172cell lines, LC3, Beclin1and ATG5mRNA and protein expression levelsignificantly increase(P <0.05) after TMZ induction, U251-MG(RNAi-STMN1) group reachstatistical highly significance (P <0.01), as well as Beclin1in A172(RNAi-STMN1).Comparing U251-MG(RNAi-STMN1)+TMZ+3-MA/Baf group andU251-MG(RNAi-STMN1)+TMZ group, the number of autophagic vacuoles reduce,achieving a statisticall significant difference (P <0.05). After the suppression of the early orlate, autophagy induced by TMZ is still enhanced. The expression level of autophagy-relatedgenes/proteins (LC3, Beclin1and ATG5) was significantly increased(P <0.05). The cellviability U251-MG(RNAi-STMN1)+TMZ+3-MA+Baf group is higher thanU251-MG(RNAi-STMN1)+TMZ group statistically (P<0.05); separated early or latesuppression of autophagy alone, comparing U251-MG(RNAi-STMN1)+TMZ+3-MA/Bafgroup and U251-MG(RNAi-STMN1)+TMZ group, the cell viability increase does not reachthe statistical significant difference (P>0.05).Conclusion: TMZ can induce autophagy in U251-MG and U251-MG (Si-STMN1),autophagy-related genes/proteins significantly increase after TMZ induction; inhibitingautophagy in early and late stage jointly, the sensitivity to TMZ of U251-MG (Si-STMN1)increase, indicating that autophagy can protect glioma cells, reducing TMZ toxicity.