The Regulatory Effects Of Mir-181a-5p On Temozolomide Resistance And Invasion Capacity In Glioblastoma Cells

Background:Glioblastoma has become a difficult problem to be solved in clinic due to its high morbidity,high recurrence rate and high mortality.Temozolomide(TMZ)is a new oral alkylating agent,which can penetrate the blood-brain barrier to tumor lesions,effectively restrain glioma cell proliferation and induce cell apoptosis,and prolong patient's survival time.However,there is frequent occurrence of chemotherapy resistance in GBM,which results in recurrence of glioma and failure of treatment.And the mechanism is still not fully understood.miRNAsis a class of non-coding RNA composed of 19 to 25 nucleotides that are newly found in eukaryotes.miRNAs participate in the biological processes of cell proliferation,differentiation,apoptosis and cycle,and play an important regulatory role in biological development and tumorigenesis.After combining miRNAs with downstream target gene 3' non-coding region,the expression of target gene protein was regulated by transcriptional inhibition or degradation of transcriptional products.A growing number of studies found that miRNAs in a variety of normal tissue and tumor tissue expression spectrum has significant differences,indicates that miRNAs for tumor classification and prediction of treatment response plays an important role.We previously analyzedthe differential expression of a panel of miRNAs between theTMZ-resistant GBM cell line and its parental cell line by miRNA microarray.We found that the expression level of mir-181a-5pwas significantly down-regulated in TMZ-resistant GBM cells.However,the regulatory role of miR-181a-5p in chemoresistance of GBM cells and its molecular mechanism have not been reported.To sum up,we speculated that mir-181a-5p may be involved in the process of the drug resistance mechanism of GBM cells.Objective:The purpose of this study was to investigate the regulatory effect of mir-181a-5p on drug resistance and invasion of GBM cells.This study might further improve the GBM chemoresistance molecular mechanism,provide experiment basis for the research on GBM chemotherapy drug resistance,also solve the problem of GBM chemoresistance and provide a new treatment strategy.Methods:1.The expression level of mir-181a-5p was detected in U251 TR,U251,primary GBM tissues and relapsedGBM tissues to clarify the correlation between miR-181a-5p and chemoresistance of GBM.2.MiR-181a-5p mimics and inhibitors were used to modulate cellular level of miR-181a-5p in GBM cells.The effect of miR-181a-5p on drug sensitivity of GBM cells was determined by using cck-8 method.3.Flow cytometry was used to detect changes in apoptosis rate of GBM cells whichwere over-expression of miR-181a-5p,to determine whether miR-181a-5p can promote the apoptosis of GBM cells.4.The effect of miR-181a-5p on the migration ability of GBM cells was observed by wound healing assay.5.The effect of miR-181a-5p on the invasion ability of GBM cells was analyzed by In vitro invasion assay,to explore whether miR-181a-5p was involved in regulating the invasion of GBM cells.Results:1.MiR-181a-5p showed low expression in GBM resistant cell lines and recurrent tumor tissues,and the differences between the two groups were statistically significant(P<0.05).2.In vitro drug sensitivity assay:The IC50 value of TMZ in the U251 TR cells transfected with miR-181a-5p were significantly decreased by 1.6-3.1-fold(P<0.001),indicating that upregulation of miR-181a-5p expression could promote the sensitivities of GBM cells to TMZ.In contrast,the IC50 values of TMZ in the U251 cells transfected with anti-miR-181a-5p were increased by 2.6-3.3-fold(P<0.001),suggesting that loss of miR-181a-5p mimics promotes drug resistance to TMZ.3.Flow cytometric analysis of apoptosis:Compared with the control group,the up-regulated expression level of miR-181a-5p could induce the increase of apoptosis rate of GBM cells,andthe difference was statistically significant(P<0.05).4.Wound healing assay and in vitro invasion assay:Compared with the control group,decreased invasion and migration activity of GBM cells in the presence of miR-181a-5p mimics,but remarkably enhanced invasion and migration activity upon anti-miR-181a-5p treatment(P<0.05).Conclusion:1.MiR-181a-5p could increase the sensitivity of GBM cells to TMZ;MiR-181a-5p can significantly inhibit the migration and invasioncapacity of GBM cells.2.MiR-181a-5p re-expression could induce cell apoptosis in GBM cells.