CAJ | 학술논문

目的探讨微小RNA(miRNA)let-7i对胶质瘤U251干细胞成熟分化的影响及其调控机制. 方法 (1)体外培养胶质瘤U251细胞,磁珠分选出CD133阳性细胞,通过合成let-7imimics(let-7i mimics组)及let-7i control (let-7i control组)转染U251干细胞,实时荧光定量PCR验证转染后let-7i表达水平,Western blotting检测U251干细胞转染前后CD133、巢蛋白(nestin)和LIN28表达水平,利用胶质纤维酸性蛋白(GFAP)免疫荧光染色U251干细胞评价其成熟分化水平.(2)LIN28 siRNA(LIN28 siRNA组)及siRNA control(siRNA control组)转染U251干细胞后,Westernblotting检测转染前后CD133和nestin表达水平.(3)利用Targetscan软件分析及荧光素酶报告系统验证LIN28是let-7i靶基因的可能性. 结果 (1)转染let-7i mimics的U251干细胞中let-7i显著过表达,为let-7i control组的17.9倍;CD133、nestin和LIN28表达水平分别是let-7i control组13.9％、43.7％和53.6％;GFAP阳性标记指数为(83.0±1.93)％,显著高于let-7i control组[(39.7±6.73)％],差异有统计学意义(P＜0.05).(2)LIN28 siRNA转染U251干细胞后CD133和nestin表达水平分别下调为siRNA control组的23.7％和37.9％.(3)Targetscan软件分析表明LIN28 3'UTR存在let-7i的配对结合位点,荧光素酶报告系统证明LIN28是let-7i的靶基因. 结论过表达let-7i可显著下调CD133及nestin的表达水平,促进胶质瘤干细胞的成熟分化,其机制可能是通过下调LIN28表达.
목적 탐토미소RNA(miRNA)let-7i대효질류U251간세포성숙분화적영향급기조공궤제. 방법 (1)체외배양효질류U251세포,자주분선출CD133양성세포,통과합성let-7imimics(let-7i mimics조)급let-7i control (let-7i control조)전염U251간세포,실시형광정량PCR험증전염후let-7i표체수평,Western blotting검측U251간세포전염전후CD133、소단백(nestin)화LIN28표체수평,이용효질섬유산성단백(GFAP)면역형광염색U251간세포평개기성숙분화수평.(2)LIN28 siRNA(LIN28 siRNA조)급siRNA control(siRNA control조)전염U251간세포후,Westernblotting검측전염전후CD133화nestin표체수평.(3)이용Targetscan연건분석급형광소매보고계통험증LIN28시let-7i파기인적가능성. 결과 (1)전염let-7i mimics적U251간세포중let-7i현저과표체,위let-7i control조적17.9배;CD133、nestin화LIN28표체수평분별시let-7i control조13.9％、43.7％화53.6％;GFAP양성표기지수위(83.0±1.93)％,현저고우let-7i control조[(39.7±6.73)％],차이유통계학의의(P＜0.05).(2)LIN28 siRNA전염U251간세포후CD133화nestin표체수평분별하조위siRNA control조적23.7％화37.9％.(3)Targetscan연건분석표명LIN28 3'UTR존재let-7i적배대결합위점,형광소매보고계통증명LIN28시let-7i적파기인. 결론 과표체let-7i가현저하조CD133급nestin적표체수평,촉진효질류간세포적성숙분화,기궤제가능시통과하조LIN28표체.
Objective To investigate the effect of MicroRNA (miRNA) let-7i on differentiation ofglioma U251 stem cells and the potential mechanisms.Methods Glioma U251 cells were cultured in vitro and CD133 positive cells were sorted by magnetic cell separation.Composite let-7i mimics and let-7i controls were transfected into U251 cells; the let-7i expression was detected by real time-PCR; the expressions ofCD133,nestin and LIN28 were detected by Western blotting; the differentiation status was assessed via glial fibrillary acidic protein (GFAP) immunocytochemistry.Composite LIN28 siRNA and siRNA control were transfected into U251 cells; the expressions of CD133 and nestin were detected by Western blotting.Targetscan software analysis and luciferase reporter system were employed to verified the possibility of LIN28 being the target gene of let-7i.Results let-7i in the U251 cells were over-expressed in group of let-7i mimics,enjoying 17.9 fold of group of let-7i control; the expressions of CD 133,nestin and LIN28 were 13.9％,43.7％ and 53.6％,separately,of group of let-7i control; GFAP positive labeling index in group oflet-7i mimics (83.0±1.93)％ was significantly higher than that of group oflet-7i control (39.7±6.73)％ (P＜0.05).The expressions of CD133 and nestin in group of LIN28 siRNA decreased to 23.7％ and 37.9％ of those in groups of siRNA control.LIN28 3'UTR existed let-7i paired binding sites and LIN28 could be the target gene of let-7i.Conclusion over-expressing let-7i could significantly down-regulate the expressions of CD133 and nestin,and enhance the glioma stem cell differentiation through the potential mechanism of down-regulating the expression of LIN28.