The Roles And Molecular Mechanism Of Temozolomide And MiRNA In Proliferation Invasion And Migration Of Glioma Cells

Objective:TMZ is a first-line chemotherapeutic agent for high-grade glioma,but the problem of drug resistance is becoming more and more prominent,this study aims to first clarify whether there are new effective targets in the anti-tumor action of TMZ in GBM.Then,the Wnt/β-catenin pathway and KLF4,which are still to be further studied in GBM,were combined with bioinformatics analysis and physiological function verification to discuss the interaction and regulation mechanism of miR-325,miR-596 and miR-32 in GBM.It is expected to provide an experimental basis for further studies on the role of miRNAs in enhancing the sensitivity of GBM to TMZ chemotherapy.1.Aim to explore whether temozolomide has an anti-glioma process through the miRNA pathway,and its new effect targets;2.To study the role and potential mechanism of miR-325 and the target gene TFRC in inhibiting the proliferation and invasion of glioma cells;3.To study the role of miR-596 and its target gene CREPT in inhibiting the malignant biological behavior of glioma cells.4.To study the role of miR-32 and its KLF4 target gene in inhibiting the malignant biological behavior of glioma cells.Methods:1.Cell transfection was performed to overexpress or silence related genes;2.RT-qPCR was performed to detect the levels of related genes;3.Western blot was performed to detect the levels of related proteins;4.MTT assay was performed to detect the viability of glioma cells;5.CCK-8 or colony formation assay were conducted glioma cell proliferation;6.Transwell was conducted to assess glioma cell invasion;7.Wound healing migration assay was performed to assess glioma cell migration;8.Bioinformatics was performed to predict the potential target genes of microRNAs;9.Dual luciferase reporter gene assay was performed to confirm the target genes of microRNAs;10.SPSS19.0 software was performed for statistical analysis of experimental data.Results:1.Temozolomide inhibits proliferation and invasion of glioma cells by inhibiting TFRC expression: Temozolomide inhibits proliferation and invasion of glioma U87 cells by inhibiting TFRC expression in a dose-dependent manner in a certain dose range,and inhibits proliferation and invasion of glioma U87 cells;Overexpression of TFRC alleviates the inhibitory effect of temozolomide on proliferation and invasion of U87 cells in glioma.TFRC silences aggravate the inhibitory effect of temozolomide on the proliferation and invasion of glioma U87 cells.2.miR-325 inhibits glioma cell proliferation and invasion by targeting TFRC: TFRC is a target gene of miR-325 and is negatively regulated by TFRC.miR-325 was down-regulated in glioma tissues and cell lines and correlated with patient survival rate.By up-regulating miR-325,TFRC expression was inhibited to inhibit proliferation and invasion of glioma U87 and U251 cells.Over-expression of TFRC promoted proliferation and invasion of glioma U87 cells and U251 cells by downregulating miR-325 expression.3.CREPT through the activation of Wnt/β-catenin pathway to promote glioma cell proliferation and invasion and as target genes of miR-596: CREPT silence suppression glioma LN-18 cells and U-138 MG of cell proliferation and invasion;CREPT target genes of miR-596 and is subject to the negative control;miR-596 regulates proliferation and invasion of glioma LN-18 cells and U-138 MG cells.CREPT in silence or miR-596 expression inhibition of Wnt/β-catenin pathway activation;The overexpression of CREPT reduced the inhibitory effect of miR-596 on the proliferation and invasion of glioma LN-18 cells and U-138 MG cells.4.miR-32 promoted the proliferation and invasion of glioma by targeting KLF4expression: miR-32 promoted the proliferation,invasion and migration of glioma U87cells;KLF4 is a target gene of miR-32 and is negatively regulated by it.KLF4 inhibited the proliferation,invasion and migration of glioma U87 cells.Silencing of KLF4 attenuates the inhibition of miR-32 down-regulation on the proliferation and invasion of glioma U87 cells.Conclusion:1.TFRC is a novel effector of temozolomide in GBM,and also a target gene of miR-325;miR-325 inhibits glioma progression by down-regulating TFRC expression.This suggests that miR-325 targets TFRC as a tumor suppressor gene.2.miR-596 inhibits the proliferation and invasion of glioma cells by targeting CREPT by inhibiting the Wnt/β-catenin pathway mediated by miR-596,and the miR-596/Wnt/β-catenin signaling axis plays an important role in the process of glioma.miR-596 inhibited proliferation and invasion of glioma cells by targeting the expression of CREPT.miR-32 promotes proliferation and invasion of glioma cells by targeting KLF4 expression.The novel target gene of miR-596 CREPT is an oncogene.The novel target gene of miR-32 KLF4 is an contra-oncogene.