CAJ | 학술논문

目的：探讨microRNA?125a（ miR?125a）对肝癌HepG2细胞增殖、凋亡及细胞周期的影响。方法采用实时荧光定量PCR（ qPCR）法检测人肝癌HepG2细胞及人正常肝细胞7702中的miR?125a水平，同时采用真核表达载体pGenesil?1质粒制备过表达miR?125a的重组质粒pGenesil?miR?125a及表达随机序列的阴性对照pGenesil?NC，根据实验设计将HepG2细胞分为3组：空白对照组、阴性对照组和过表达组，其中空白对照组未进行转染，阴性对照组和过表达组分别成功转染pGenesil?NC和pGenesil?miR?125a，待3组转染24、48、72及96 h后采用qPCR法检测各组的miR?125a水平，噻唑盐比色法检测各组转染24、48、72及96 h的细胞增殖率，分别采用Hoechst染色和流式细胞术检测转染24、48 h后的凋亡指数和caspase?3活化率来评价凋亡情况，流式细胞仪Annexin V?FITC／PI双染流式细胞术检测各组转染48 h后的细胞周期，同时采用免疫印迹法检测转染48 h后对凋亡相关基因（ Bcl?2、Bax和Cleaved caspase?3）表达的影响。结果 HepG2细胞中miR?125a水平为（0?24±0?06），低于正常肝细胞7702细胞（P＜0?05）；与阴性表达组相比，过表达组转染24、48、72及96 h的miR?125a水平升高，增殖率降低，差异有统计学意义（ P＜0?05）；与其余两组相比，过表达组转染后的凋亡指数、caspase?3活化率、G0／G1期细胞比例及Bax和Cleaved caspase?3水平均升高，S和G2／M期细胞比例及Bcl?2水平均降低，以上差异有统计学意义（ P＜0?05）。结论肝癌HepG2细胞中miR?125a低表达，且过表达miR?125a可抑制增殖、诱导凋亡及G0／G1期细胞阻滞，在肝癌的发生发展中可能起到抑癌基因的作用。
목적：탐토microRNA?125a（ miR?125a）대간암HepG2세포증식、조망급세포주기적영향。방법채용실시형광정량PCR（ qPCR）법검측인간암HepG2세포급인정상간세포7702중적miR?125a수평，동시채용진핵표체재체pGenesil?1질립제비과표체miR?125a적중조질립pGenesil?miR?125a급표체수궤서렬적음성대조pGenesil?NC，근거실험설계장HepG2세포분위3조：공백대조조、음성대조조화과표체조，기중공백대조조미진행전염，음성대조조화과표체조분별성공전염pGenesil?NC화pGenesil?miR?125a，대3조전염24、48、72급96 h후채용qPCR법검측각조적miR?125a수평，새서염비색법검측각조전염24、48、72급96 h적세포증식솔，분별채용Hoechst염색화류식세포술검측전염24、48 h후적조망지수화caspase?3활화솔래평개조망정황，류식세포의Annexin V?FITC／PI쌍염류식세포술검측각조전염48 h후적세포주기，동시채용면역인적법검측전염48 h후대조망상관기인（ Bcl?2、Bax화Cleaved caspase?3）표체적영향。결과 HepG2세포중miR?125a수평위（0?24±0?06），저우정상간세포7702세포（P＜0?05）；여음성표체조상비，과표체조전염24、48、72급96 h적miR?125a수평승고，증식솔강저，차이유통계학의의（ P＜0?05）；여기여량조상비，과표체조전염후적조망지수、caspase?3활화솔、G0／G1기세포비례급Bax화Cleaved caspase?3수평균승고，S화G2／M기세포비례급Bcl?2수평균강저，이상차이유통계학의의（ P＜0?05）。결론간암HepG2세포중miR?125a저표체，차과표체miR?125a가억제증식、유도조망급G0／G1기세포조체，재간암적발생발전중가능기도억암기인적작용。
Objective To explore the influence of microRNA?125a( miR?125a) on the proliferation, apoptosis and cell cycle of human hepatoma HepG2 cells. Methods The real?time fluorescence quantitative PCR( qPCR) was employed to measure the miR?125a level in human hepatocellular carcinoma HepG2 cell and normal liver 7702 cells. The eukaryotic expression plasmid vector pGene?sil?1 was used to construct recombinant plasmid pGenesil?miR?125a. Meanwhile, a control plasmid pGenesil?control with random se?quence was constructed. According to the experimental design, the HepG2 cells were divided into 3 groups: blank control group ( HepG2 cells without transfection ) , negative control group ( HepG2 cells transfected with pGenesil?NC ) and over?expression group ( HepG2 cells transfected with pGenesil?miR?125a) . The qPCR was used to investigate the miR?125a level of three groups at 24, 48, 72 and 96 h after transfection. Tetrazolium salt( MTT) colorimetric assay was used to assess the proliferative responses of HepG2 at 24, 48, 72 and 96 h after transfection. The Hoechst staining and flow cytometry were used to evaluate the apoptosis index and caspase?3 ac?tivation rate to evaluate the apoptosis at 24 and 48 h after transfection. The cell cycle at 48 h after transfection was determined by An?nexin V?FITC/PI double staining with the use of flow cytometry analysis. Western blotting was employed to analyze the protein levels of apoptosis related genes( Bcl?2, Bax and Cleaved caspase?3) at 48 h after transfection. Results The level of miR?125a in HepG2 cell was(0?24±0?06), lower than that of 7702 cell(P<0?05). MiR?125a level increased but proliferation rate decreased at 24, 48, 72 and 96 h after transfection in over?expression group versus negative control group with statistically significant difference( P<0?05) . In con?trast to other two groups, there were elevated apoptosis index, caspase?3 activation rate, percentage of G0/G1 and protein level of Bax and Cleaved caspase?3 but decreased percentage of S and G2/M phase and protein level of Bcl?2 with statistically significant difference ( P<0?05) . Conclusion There is low expression of miR?125a in HepG2 cells, and the over?expression of miR?125a can inhibit the proliferation, induce cell apoptosis and G0/G1 arrest, playing as tumor suppressor gene in the development of hepatocellular carcinoma.