CAJ | 학술논문

目的探讨miR - 203靶向抑制survivin对人胶质瘤细胞株U251生长和增殖的影响.方法利用生物信息学预测miR -203靶基因；通过脂质体将miR - 203模拟物或表达质粒转染至U251细胞,使用Real - time PCR和Western blot检测靶基因survivin的mRNA和蛋白水平,采用双荧光素酶报告基因系统进行功能验证,应用细胞生长曲线、MTT实验、流式细胞术评价细胞生长、增殖和凋亡的变化及对肿瘤坏死因子相关凋亡诱导配体(TRAIL)和塞来昔布的敏感性.结果 TargetScan预测发现survivin是miR - 203的靶基因；转染miR - 203模拟物或表达质粒后,Real - time PCR与Western blot结果分别提示survivin的mRNA和蛋白水平下调(P＜0.05)；双荧光素酶报告分析技术证实抗凋亡基因survivin为miR - 203的直接靶点；细胞生长曲线结果显示miR - 203组的生长速度明显受抑(P＜0.05)；MTT实验表明miR - 203组的增殖能力减弱(P＜0.05)及药物敏感性增加；流式细胞术结果示miR -203组的凋亡比例明显升高(P＜0.01).结论 miR - 203在胶质瘤细胞中能靶向抑制survivin的表达,是一个抗增殖、促凋亡的miRNA,有可能作为今后胶质瘤干预治疗的新靶点.
목적 탐토miR - 203파향억제survivin대인효질류세포주U251생장화증식적영향.방법 이용생물신식학예측miR -203파기인；통과지질체장miR - 203모의물혹표체질립전염지U251세포,사용Real - time PCR화Western blot검측파기인survivin적mRNA화단백수평,채용쌍형광소매보고기인계통진행공능험증,응용세포생장곡선、MTT실험、류식세포술평개세포생장、증식화조망적변화급대종류배사인자상관조망유도배체(TRAIL)화새래석포적민감성.결과 TargetScan예측발현survivin시miR - 203적파기인；전염miR - 203모의물혹표체질립후,Real - time PCR여Western blot결과분별제시survivin적mRNA화단백수평하조(P＜0.05)；쌍형광소매보고분석기술증실항조망기인survivin위miR - 203적직접파점；세포생장곡선결과현시miR - 203조적생장속도명현수억(P＜0.05)；MTT실험표명miR - 203조적증식능력감약(P＜0.05)급약물민감성증가；류식세포술결과시miR -203조적조망비례명현승고(P＜0.01).결론 miR - 203재효질류세포중능파향억제survivin적표체,시일개항증식、촉조망적miRNA,유가능작위금후효질류간예치료적신파점.
Objective To investigate the influence of miR -203 on growth and proliferation of human glioma cell line U251.Methods Target of miR - 203 was forecasted by bioinformatics.U251cells were transfected with mimics or expression plasmid of miR - 203 using Lipofectamine 2000. After transfection,mRNA and protein level of target gene survivin was detected by Real -time PCR and Western blot.The ability of miR - 203 was confirmed with dual - luciferase activity assay.Cell growth,proliferation apoptosis and sensitivity to TRAIL and celecoxib were determined by growth curve,MTT assay and flow cytometry.Results Predicting outcomes of TargetScan indicated that survivin was target of miR - 203.After over - expression of miR - 203,Real - time PCR and Western blot showed that mRNA and protein level of survivin were down - regulated,respectively ( P ＜ 0.05 ). Dual - luciferase activity assay demonstrated that survivin was direct target of miR -203.Growth curve and MTT assay revealed that growth and proliferation ability of miR - 203 group were significantly suppressed ( P ＜ 0.05 ),and sensitivity to TRAIL and celecoxib was increased.U251cells transfected miR -203 had a higher proportion of apoptosis than control group ( P ＜0.01).Conclution miR- 203 is an "anti -proliferation and pro- apoptosis"miRNA which can specifically inhibit survivin expression.In the future,miR -203 may be an attractive target for therapeutic intervention in glioma.