| Temozolomide(TMZ)with radiotherapy is the current standard of care for newly diagnosed glioma.However,glioma patients who are treated with the drug often develop resistance to it and some other drugs.Recently studies have shown that microRNAs(miRNAs)play an important role in drug resistance.In present study,we first examined the sensitivity to temozolomide in six glioma cell lines,and established a resistant variant,U251MG/TR cells from TMZ-sensitive glioma cell line,U251MG.We then performed a comprehensive analysis of miRNA expressions in U251MG/TR and parental cells using cancer microRNA PCR Array.Among the downregulated microRNAs was miR-16,members of miR-15/16 family,whose expression was further validated by qRT-PCR in U251MG/TR and U251MG cells.The selective microRNA,miR-16 mimics or inhibitor was respectively transfected into U251MG/TR cells and AM38 cell.We found that treatment with the mimics of miR-16 greatly decreased the sensitivity of U251MG/TR cells to temozolomide,while sensitivity to these drugs was increased by treatment with the miR-16 inhibitor.In addition,the downregulation of miR-16 in temozolomide-sensitive AM38 cells was concurrent with the upregulation of Bcl-2 protein.Conversely,overexpression of miR-16 in temozolomide-resistant cells inhibited Bcl-2 expression and decreased temozolomide resistance.In conclusion,MiR-16 mediated temozolomide-resistance in glioma cells by modulation of apoptosis via targeting Bcl-2,which suggesting that miR-16 and Bcl-2 would be potential therapeutic targets for glioma therapy.PART 1.Establishment of temozolomide-resistant U-251MG cell lineObjective:To establish the temozolomide-resistant cell line,which has significant value for the study of molecular mechanism of drug-resistance and find out the strategy to reverse gliomas drug-resistance.And view the characterization of the drug-resistance cell line and verify the important role of related genes in the drug resistance mechanism.Methods:Human glioblastoma cell lines were cultured with 10%fetal bovine serum,100 units/ml penicillin,and 0.1 mg/ml streptomycin in 5%CO2 atmosphere at 37 ?.Cells were seeded into 96-well culture plates and incubated at 37 ?.Temozolomide was dissolved in DMSO.After treatment with different concentrations of temozolomide for 72 h,20 ?L CCK-8 reagent was added to each well and incubated for2 h at 37 C and the optical density(OD)was measured by a microplate reader at 490 nm.Each temozolomide concentration was tested in triplicate in 96-well plates,and experiments were repeated independently at least three times.The 50%inhibitory concentration(IC50)was calculated with GraphPad Prism software using the sigmoidal dose-response function.Then we chose the U251/MG.Chemotherapeutic resistance in U251MG human glioma cells was initiated by the stepwise revulsion with TMZ.The cell morphology of U251MG and U251MG/TR was observed under optical microscope.The drug resistance was detected by CCK-8.Cell apoptosis was tested by flow cytometry.Result:To determine whether temozolomide was associated with chemoresistance of glioma cells,CCK-8 assay was performed to detect temozolomide sensitivity in six glioma cell lines.Two of them LN382,and U138MG,exhibited a higher IC 50 than the other four cell lines A-172,AM-38,U-251MG and KMG4.Next,U251MG wild type(U251Wt)cells were exposed to 100 ?M TMZ for 2 weeks to generate TMZ-resistant variant.The majority of the cells died,but a small population survived and propagated.We then selected surviving colonies and established U251MG TMZ-resistant cells(U251MG/TR).IC50 for the growth inhibition of temozolomide to U-251MG and U-251MG/TR are 25.3 mM and 151.6 mM,respectively.Furthermore,flow cytometry was used to detect cells apoptosis of U-251MG and U-251MG/TR cells after temozolomide treatment(50 ?M),and showed that apoptosis of U-251MG significantly increased compared with control cells,But the correspondingly converse results was observed in U-251MG/TR cells.Conclusion:A stable drug-resistant cell line U251/TMZ induced by TMZ was established successfully in 6-month culture,meanwhile,increasing the concentration of TMZ stepwisely was the important strategy.PART 2.Study on relationship between MIR-16 and U251MG/TRObjective:MIR-16 was a closely related to the development of a variety of malignant tumors.But the relationship with glioma resistance to chemotherapy was unclear.We discuss on the relationship between them to explore the role of MIR-16 play in the mechanism of drug resistance of gliomas cell.Methods:The expression profile of 88 cancer-related miRNAs was determined using a 96-well plate cancer RT2 miRNA PCR array in an in vitro cell culture model composed of glioma cell line U251MG and U251TMZ-resistant(U251MG/TR)cells.Total RNA was extracted using the TRIzol reagent and reverse transcribed using the RT2 miRNA.The resulting cDNA was then diluted,mixed with 2ŚRT2 SYBR Green PCR Master Mix,and loaded into the wells of a PCR array plate to allow real-time PCR amplification and detection.Data analysis was performed with the Web-based software package for the miRNA PCR array system.Result:To identify miRNAs specifically deregulated in U251MG/TR cells,we performed a comprehensive analysis of miRNA expression in U251MG and U251MG/TR cells using cancer microRNA PCR Array.Twelve miRNAs were overexpressed(>2.0-fold)and eight were under expressed(<2.0-fold)in U251MG/TR cells compared to U251MG cells.To validate the microRNA PCR Array data,we utilized TaqMan real-time RT-PCR assay for miR-150,let-7a,miR-9,miR-16,miR-98 and miR-125b,the three most up-regulated miRNAs and the three most down-regulated miRNAs,these miRNAs were certainly up-regulated and down-regulated in U251MG/TR cells,suggesting that deregulation of those miRNAs is involved in the acquisition of TMZ resistance in GBM cells.From the list of differentially expressed LncRNAs,we focused on MIR-16,as previous reports showed that it was related with resistance.To identify the effect of MIR-16 on TMZ sensitivity in U251MG cells,we examined the mRNA expression of MIR-16 in these cell lines firstly.The correlation between the IC 50 values and the relative mRNA expression of miR-16 was analyzed.The IC 50 values of temozolomide significantly correlated with the expression level of miR-16 in these glioma cells.Furthermore,we performed CCK-8 assay to detect the cell viability of AM38 cells transfected with MIR-16 inhibitor or U251MG/TR cells transfected with MIR-16 mimics for 24 h,respectively,followed by treatment with TMZ for 48 h.Results showed that overexpression of miR-16 rendered U251MG/TR cells more resistant to temozolomide.U251MG/TR cells transfected with vector control had an IC 50 value of 38.2 ?M;whereas the IC50 value of U251MG/TR cells overexpressed with MIR-16 was 93.6 ?M.Instead,transfection of MIR-16 inhibitor significantly increased TMZ resistance in AM38cells(IC50control:151.6 ?M;IC50AM38/miR-16inhibitor:50.4 ?M,).In summary,these results suggest that miR-16 contribute to TMZ resistance in glioma cells.Conclusion:The expression of MIR-16 was significantly related to temozolomide-resistance.Transfection of MIR-16 mimics into the U251MG/TR could significantly improve the cell inhibition rate of temozolomide,showed that it could regulated the chemosensitivity of gliomas.PART 3.The mechanisms of MIR-16 regulate on the chemotherapy sensitivity of U251MG/TRObjective:To study on the effect of MIR-16 regulate on the chemotherapy sensitivity of U251MG/TR cell line,analysis the expression chages of drug-resisitance gene BCL-2 and detect the activation of the signaling pathway after MIR-16 transfected into gliomas drug-resistance cell line,and to explore the possible mechanisms of MIR-16.Methods:The extent of apoptosis was determined by the flow cytometric measurement through Annexin V-FITC apoptosis detection kit.Cells treated as above described.After 4d,cells were harvested and washed twice with cold PBS.Then,cells were stained in 1 mL Annexin V binding buffer with 10 ?L of PI solution and 5 ?L of Annexin V-FITC for 10 min at RT and analyzed by flow cytometry.Cells were harvested and homogenized with cell lysis buffer.Then,the homogenates were centrifuged for 30 min at 4 ?,12000 rpm,and the supernatants were collected as protein samples.Protein amounts were measured.Equal amounts of protein samples were separated by denaturing 10%SDS-PAGE and transferred onto polyvinylidenedifluoride membranes.After transfer,membranes were incubated in blocking solution,probed with various antibodies,washed,and visualized using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence reagents.Result:To determine the mechanisms by which MIR-16 decreased resistance of glioma cells to temozolomide,we analyzed the effect of MIR-16 low expression on temozolomide induced apoptosis.AM38 cells stably transduced with MIR-16 were treated with 50?M temozolomide for 6 h,and then cells were washed twice and placed in temozolomide free medium to grow for 48 h.Cell apoptosis was determined by AnnexinV/PI fowcytometry assays.The apoptosis rate of AM38 cells group and MIR-16inhibitor group were 3%and 6%,respectively.It suggests that low expression of MIR-16 inhibits temozolomide induced apoptosis.To study underlying molecular mechanisms by which MIR-16 inhibits temozolomide induced apoptosis,we analyzed several apoptosis related proteins and found Bcl-2 was significantly upregulatedin AM38 cells stably transduced with MIR-16.But the correspondingly converse results was observed inU251MG/TR cells.Taken together,these data suggest that MIR-16 inhibits temozolomide-induced apoptosis via upregulation of Bcl-2 in glioma cells.Conclusion:The mechanism responsible for resistance of glioma cells to temozolomide was associated with miR-16-mediated downregulation of Bcl-2.MiR-16 may function as an important modifier of the response of glioma cells totemozolomide.A new strategy combining current regimens with compounds targeting miR-16 may significantly improve the therapeutic outcome of temozolomide-resistant glioma. |
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