The Effects Of MicroRNA-519a In Biological Behavior And Temozolomide Chemosensitivity Of GBM Cells

Glioblastoma(GBM)is the most common and lethal cancer in the central nervous system.Contributing to its poor prognosis are numerous therapeutic challenges including aggressive growth rates,tumor heterogeneity,radiochemotherapy resistance and the presence of blood-brain barrier.Currently,GBM has a 5-year survival rate of only 10%and a median survival time of 15 months following treatment of postoperative radiotherapy combined with adjuvant temozolomide(TMZ)chemotherapy.At present,GBM research is more and more focused on molecular biology mechanism research,in order to find a new target that can effectively treat GBM.MicroRNAs(miRNAs)are a class of non-coding small-molecule single-stranded RNA.In recent years,many studies have shown that abnormalities in the expression of miRNAs are related to the development of glioma.miRNAs have been found to contribute to sustained proliferative signaling and evasion of growth suppressors,resistance to cell death,differentiation,and apoptosis,enabling of replicative immortality,induction of angiogenesis,and activation of invasion and metastasis.More importantly,miRNAs are also important regulators of drug resistance in GBM.Autophagy,also known as type Ⅱ cell programmed death,refers to the biological processes in which cells transport their own cytoplasmic substances into lysosomal degradation and reuse.As a way of programmed cell death,its impact on the sensitivity of GBM chemotherapy is still controversial.Studies have shown that TMZ can promote autophagy of GBM cells and also shows that autophagy may have a dual role.The specific mechanism of autophagy in the role of chemotherapy has not yet clear.Our previous studies have found that miR-517c,as a member of the C19MC(The chr19q13.41 microRNA cluster),can inhibit autophagy and epithelial-to-mesenchymal(-like)transition phenotype in human GBM via disturbing p53 nuclear translocation conducted by KPNA2.What is the biological effect of miR-519a as a member of the C19MC in GBM?Is it the same as the function of miR-517c involved in autophagy regulation?Whether it is also involved in GBM resistance to TMZ chemotherapy?These questions are worthy of further study.This study continued to explore the biological effects of miR-519a on GBM and the specific molecular mechanisms;we alsofocused onthe effect of miR-519a on chemosensitivity and the relationship with autophagy;Aim to seek a new strategy for the treatment of GBM.The role of microRNA(miRNA)in the tumorigenesis has been extensively studied and it sheds light on the potential application as an important adjuvant treatment since the beginning of this century.In our previous work,the chr19q13.41 microRNA(miRNA)cluster(C19MC)that encodes 54 microRNAs was discovered with high DNA copy number in the 11/45 cases of supratentorial primitive neuroectodermal tumors(sPNET).Expression profiles demonstrate significant enrichment of self-renewal and survival genes in C19MC amplified tumors.Among the microRNAs,miR-512-3p,517a,517c,519a,and 520g has significantly higher expression in sPNET samples with C19MC amplification.Moreover,these miRs expression data in two neural stem cell,different parts of fetal brain and Adult brain hinted miR-517c and 519a might play important biological function in embryonic development.In order to test if the miRs also play some biological function in GBM cells,six cell lines of GBM were selected to undergo the Realtime-qPCR.Results indicated that the miR-517c and 519a have higher expression level in two cell lines The above data promoted us to do further investigation.In this study,we sought to comprehensive analyze the precise biological function of miR-517c in GBM.Whether the function of miR-517c is related to autophagy and EMT?Whether the biological function is dependent on p53 phenotype in GBM cells in vitro and in vivo?Aim to find a new strategy for treating GBM.Chapter 1 MiR-519a functions as a tumor suppressor in glioma by targeting the oncogenic STAT3 pathwayPurpose:To investigate the effect of miR-519a on the malignant biological behavior of GBM and its mechanism.Methods:qRT-PCR was used to detect the expression of miR-519a in GBM cell line and tumor tissue.The survival analysis of GBM patients in our unit and TCGA database was further investigated by kaplan-meier method.We transfected miR-519a mimics into GBM cell line and primary cells to observe the effects of miR-519a on cell proliferation,apoptosis,migration and invasion.Using the bioinformatics method to predict the target gene and then using double luciferase reporter gene system to verify the predicted target sequence.qRT-PCR,Western-blot and immunohistochemistry were used to verify STAT3 as the target gene for miR-519a at the cellular and tumor tissue levels.In the GBM cell line,the pCDNA3.1-STAT3 plasmid and miR-519a mimics were co-transfected to confirm whether overexpression of STAT3 could reverse the effect of miR-519a on cell proliferation,apoptosis,migration and invasionResults:We demonstrated that miR-519a expression was frequently downregulated in GBM specimens and cell lines,and that low-levels miR-519a expression significantly correlated with poor outcomes associated with GBM.Analysis of The Cancer Genome Atlas also demonstrated that low miR-519a expression can predict poor clinical outcomes in classical and proneural GBM subtypes.Functionally,re-expression of miR-519a effectively reduced GBM cell proliferation,migration,and invasion.Mechanistically,we confirmed that the signal transducer and activator of transcription 3(STAT3)3’-UTR was a putative target of miR-519a,and that re-expression of STAT3 abrogated miR-519a function in GBM cells.Furthermore,we found that STAT3 expression negatively correlated with that of miR-519a in human GBM tissues.Conclusion:These results elucidated the prognostic value and tumor-suppressor role of miR-519a in GBM and further suggested it as a potential therapeutic target for GBM treatment.Chapter 2MiR-519a enhances the sensitivity of GBM cells to temozolomide by inhibiting STAT3 to promote autophagyPurpose:To investigate the chemosensitivity of miR-519a to enhance glioma cells and its mechanism.Methods:By gradually increasing the concentration of Temozolomide(TMZ),we obtained the the U87-MG/TMZ cell line which was resistant to TMZ.Apply MTT to observe vitality of the cells when U87-MGand U87-MG/TMZ had been acted on for different time by different density of TMZ.qRT-PCR was used to detect and compare the expression of miR-519a in U87-MG cells and U87-MG/TMZ cells.After changes in miR-519a expression in U87-MG and U87-MG/TMZ cells,MTT,apoptosis and clone formation experiments were performed to detect the changes in TMZ sensitivity.The autophagy levels of U87-MG and U87-MG/TMZ cells were detected by transmission electron microscopy,LC3B protein immunofluorescence and Western blot.After respectively transfected with miR-519a mimics and miR-519a inhibitor into U87-MG/TMZ and U87-MG cells,we added a specific concentration of TMZ to the cell culture medium and detected the changes of autophagy levels.After altering the autophagy level of the cells U87-MG/TMZ and U87-MG cells respectively transfected with miR-519a mimics and miR-519a inhibitor,we added a specific concentration of TMZ to the cell culture medium and applied MTT,apoptosis and clonal formation experiments to detect the sensitivity of cells to TMZ.The STAT3/Bcl-2/Beclinl pathway levels in U87-MG cells and U87-MG/TMZ cells were detected by qRT-PCR.After respectively co-transfected miR-519a inhibitor,siSTAT3 and miR-519a mimics,pCDNA3.1-STAT3 in U87-MG and U87-MG/TMZ cells,we added a specific concentration of TMZ to the cell culture medium and applied western blotting to detect the expressions of STAT3、Bcl-2、Beclin 1、Bax and Caspase 3.To establish a nude mouse subcutaneous transplanted tumor model to detect miR-519a on GBM chemotherapy sensitization.The expression of LC3,p-Stat3(Y705),Bax,Caspase-3 and Bcl-2 protein were detected by western-blot and immunohistochemistry.The expression of autophagy-related vesicles in each group was detected by transmssion electron microscopy.Results:Compared with U87-MG,the expression level of miR-519a in U87-MG/TMZ cells decreased,and miR-519a could increase the sensitivity of GBM cells to temozolomide;Overexpression or inhibition of intracellular miR-519a levels can enhance or decrease the sensitivity of GBM cells to temozolomide,respectively;MiR-519a induced more autophagy on the basis of TMZ,and autophagy also mediated the chemosensitivity of miR-519a to GBM;The mechanism of miR-519a inducing autophagy to promote apoptosis is to inhibit the expression of autophagy-related protein Beclin 1 by inhibiting the expression of STAT3.Animal level further confirmed the chemosensitivity of miR-519a to GBM and the effect of miR-519a on autophagy and apoptosis.Conculsion:MiR-519a increased the sensitivity of GBM cells to TMZ.Autophagy mediates the effect of miR-519a on chemosensitivity of GBM cells.MiR-519a induced autophagy by inhibiting STAT3/Bcl-2/Beclinl pathway.