Up-regulation Of MiR-497 Confers Resistance To Temozolomide In Human Glioma Cells By Targeting MTOR/Bcl-2

Human gliomas are the most frequent and most aggressive primary brain tumor that exhibits malignant aggressiveness and poor prognosis.Currently,although surgical operation combined with radiotherapy and chemotherapyare effective remedies for the treatment of primary glioma,the prognosis of glioma patients still remains poor,with a median survival time of about 12 months.TMZ is a secondgeneration of alkylating agent that induces apoptosis via methylation of guanine residues.TMZ concurrently and after radiotherapy significantly improves the overall survival compared with radiation therapy alone and has the advantage of wide applicability and minimal additional toxicity.It has become the current standard chemotherapeutic drug in the treatment for glioma.However,the development of inherent or acquired TMZ-resistance limits its clinical application,and the mechanisms of TMZ-resistance are not fully understood.Thus,identification of new molecular targets is urgently required to fight against TMZ-resistance in glioma.MicroRNAs(miRNAs)are a class of endogenous and small non-coding RNAs with 21~23 ribonucleotides,which can induce m RNA degradation or act as repressors of translation through directly binding to a target site in the 3′-untranslated regions(UTRs)of their target genes.Existing studies found that miRNA-mediated gene regulation involved in multiple pathological processes including cell proliferation,differentiation,migration,survival,and tumorigenesis.Recent evidence has shown that miRNAs also plays an vital role in chemotherapeutic resistance.For instance,miR-873 was found to improve the chemo-sensitivityof glioblastoma cells to cisplatin by targeting Bcl-2.MiR-16 was reported to mediate TMZ-resistance in glioma cells by modulation of apoptosis,highlighting miRNAs as potent chemo-resistant modulators in the treatment of glioma.MiR-497 is found as a tumor suppressive miRNA in most human cancer.Such function is exerted partly by its anti-proliferative and anti-growth potential.Previous study showed that miR-497 induced breast cancer cell apoptosis by negatively regulating Bcl-2 protein expression.Recent studies found that overexpression of miR-497 decreases cisplatin resistance of ovarian cancer cells in vitro and in vivo.However,whether miR-497 regulates other target genes to mediate glioma cell growth is still unknown,and the relationship between miR-497 and chemotherapeutical resistance in glioma cells is not fully elucidated.This study was designed to investigate the molecular mechanisms of miR-497 in TMZ-resistant glioma.Our research will be discussed from the following four parts.1.Establishment and characterization of the drug-resistance glioma cell line.Objective: Analyze the sensitive of TMZ in several glioma cell lines.Establish the drug-resistance human glioma cell line induced by TMZ,which has significant value for the study of molecular mechanism of drug-resistance and find out the strategy to reverse gliomas drug-resistance.Methods: Analyze the half maximal inhibitory concentration(IC50)in several giloma cell lines(SF295,SHG-44,U138,LN382,U87 and U251).To establish the TMZ-resistant glioma cell lines,glioma cells were exposed to gradually increasing concentrations of TMZ(5 μmol/L-100 μmol/L)in culture media for 6 months.The cell morphology of glioma cells was observed under optical microscope.CCK-8 assay was used to calculate the IC50 and resistance index(RF).Results: The values of IC50 in several giloma cell lines(SF295,SHG-44,U138,LN382,U87 and U251)were 54.8,149.6,4 96.1,349.2,9 4.8 and 264.8 u M/L,respectively.Our results showed that SF295 and U87 exhibited a lower IC50 than the other cell lines,were chose in the subsequent experiments.We successfully established TMZ resistant cell lines SF295/TR and U87/TR in six months.The resistance phenotype was stable and cell autonomous,as it was not reversed by culturing U87-TR or SF295-TR cells in TMZ-free medium for up to 6 months.Optical microscopy showed that the volume of drug-resistant cells was increased and irregular shaped nucleus obviously increased,intracytoplasmic granular material increased.These U87-TR and SF295-TR cell lines are approximately 6 times resistant to TMZ compared with their parental cell lines,respectively.Conclusions:Two stable TMZ-resistant glioma cell lines U87-TR and SF295-TR were induced by the stepwise revulsion with TMZ successfully in six months,increasing the concentration of TMZ stepwisely was the vital strategy.2.Role of miR-497 in drug-resistant of glioma cells in vitro.Objective: Analyze the differences of miR-497 expression in the resistant and sensitive glioma cells.Gain-of-function and loss-of-function assays were performed to evaluate the anti-proliferative and pro-apoptotic effects of miR-497 on glioma cells in vitro.We aim to determine the role of miR-497 in drug-resistant glioma cells in vitro.Methods: qRT-PCR was used to determined the relative level of miR-497 in a panel of glioma cell lines,namely SF295,SHG-44,U138,LN382,U87 and U251,along with NHA,which is a normal human astrocytes.Analyze the level of miR-497 in U87-TR and SF295-TR cell lines as well as their matched parental cell lines.U87-TR and SF295-TR cells with high endogenous miR-497 levels were transfected with anti-miR-497 or anti-miR-NC.In addition,U87 and SF295 cells were transfected with miR-497 or negative control.The cellular proliferation was detected by Cell Counting Kit-8(CCK-8)assay.The apoptosis of glioma cells was quantified by DAPI staining under fluorescence microscopy.Results:The results demonstrated that the expression of miR-497 was increased in all the six glioma cell lines than that in NHA.Interestingly,the abundance of miR-497 was positively correlated with IC50 values in glioma cell lines.Investigations into such information indicated that miR-497 was closely associated with TMZ sensitivity in glioma cells.Furthermore,our results shown that the level of miR-497 was up-regulated in U87-TR and SF295-TR cell lines compared with their matched parental cell lines.The expression of miR-497 was decreased significantly after the glioma cells were transfected with anti-miR-497.Moreover,transfection of miR-497 led to a significant increase in its expression.Additionally,the result of CCK-8 assay indicated that the down-regulation of miR-497 significantly reduced U87-TR and SF295-TR cells resistance to TMZ.On the contrary,the up-regulation of miR-497 dramatically increased U87 and SF295 cells tolerance to TMZ.Our data showed clearly that the inhibition of miR-497 promoted apoptosis induced by TMZ in U87-TR cells.However,the ectopic expression of miR-497 prevented the percentage of apoptotic cells in SF295 cells induced by TMZ.(1)MiR-497 was closely associated with TMZ sensitivity in glioma cells.(2)The level of miR-497 was up-regulated in TMZ-resistant cell lines compared with their matched parental cell lines.(3)The inhibition of miR-497 promoted apoptosis induced by TMZ in U87-TR cells,whereas the ectopic expression of miR-497 prevented the percentage of apoptotic cells in SF295 cells induced by TMZ.3.mTOR/Bcl-2 are the potential targets of miR-497.Objective: To investigate the potential target of miR-497 and explore the role of targets in the biological mechanisms of drug-resistance in glioma cells.We open up new areas for exploration the effective reversal of tumor resistance.The research on miR-497 and its potential target will provide new ideas and methods for the clinical treatment of glioma in the future.Methods: Bioinformatic analyses were used to predict the target genes hsa-miR-497.Western blot was used to detected target protein expression in glioma cells in vitro.The cellular proliferation was detected by CCK-8 assay.The apoptosis of glioma cells was quantified by DAPI staining under fluorescence microscopy.Results: Our results demonstrated that human mTOR and Bcl-2 m RNA contains a binding site for miR-497.The silencing of miR-497 decreased the protein levels of both mTOR and Bcl-2.Whereas the levels of endogenous mTOR and Bcl-2 were increased after glioma cells were transfected with miR-497.Furthermore,the knockdown of mTOR and Bcl-2 exerted a similar effect as depletion of miR-497 on reducing both glioma cell lines resistance to TMZ treatment.In addition,our results showed that the loss of mTOR and Bcl-2 increased the apoptotic cells induced by TMZ in U87-TR cells.Conclusions:(1)The levels of mTOR and Bcl-2 were positively regulated by miR-497.(2)The up-regulation of miR-497 promotes acquisition of TMZ-resistant ability in glioma cell by inducing mTOR and Bcl-2 over-expression.4.Role of miR-497 in drug-resistant of glioma cells in vivo.Objective: To determine whether miR-497 influences drug-resistant of glioma cells in vivo,and explore the possibilities of miR-497 as a therapeutic target in the treatment of Conclusions: drug-resistant glioma.Methods: U87/TR cells stably transfected with lenti-anti-NC or lenti-anti-miR-497 were constructed.Then the glioma subcutaneous xenograft tumor model was established to compare the effect of TMZ on U87-TR/lenti-anti-NC or U87-TR/lenti-anti-miR-497 cells formed tumors.Furthermore,the immunohistochemical detection of cell proliferation marker Ki-67 expression in the tumor tissues was performed.In addition,quantitative data showed that Tunel staining of these tissue sections indicated significant differences in the percentage of Tunel-positive cells in the tumors derived from the anti-miR-497 + TMZ group compared with the anti-NC + TMZ group.Western blot was used to detected the levels of mTOR and Bcl-2 protein in glioma cells in vivo.Results: The xenograft tumor weights were indicative of a significant decrease in the U87-TR/lenti-anti-miR-497 + TMZ(anti-miR-497 + TMZ)group in comparison with U87-TR/lenti-anti-NC + TMZ(anti-NC + TMZ)group.The level of miR-497 was down-regulated in anti-miR-497 + TMZ group tumor tissues than that in anti-NC + TMZ group tumor tissues.The percentage of Ki-67 positive stained cells was significantly higher in the tumor derived from anti-NC + TMZ group than its expression in the anti-miR-497 + TMZ group.In addition,Tunel staining of these tissue sections indicated significant increase in the percentage of Tunel-positive cells in the tumors derived from the anti-miR-497 + TMZ group compared with the anti-NC + TMZ group.Subsequently,our results demonstrated the expression of mTOR and Bcl-2 were markedly decreased in U87-TR/lenti-anti-miR-497 cells formed tumors compared with that in U87-TR/lenti-anti-NC cells formed tumors at the translational level.Conclusions: Our in vivo findings provides promising evidence supporting miR-497 as a valuable future target for glioma treatment,and miR-497 might be used as a “supplement” in TMZ-based chemotherapy.In conclusion,our results are consistent with the hypothesis that miR-497 as a glioma promoter,is significantly correlated with the TMZ-resistance of human glioma cells both in vitro and in vivo.Moreover,mTOR and Bcl-2 were identified as potential physiological targets of miR-497 with biological function that are implicated in the development of TMZ-resistance phonotype.This work highlight the therapeutic potential of miR-497 in the functional regulation of glioma.