慢病毒介导微小RNA-20a过表达抑制胰腺癌细胞的增殖

Inhibitory effects of ientivirus-mediated overexpression of microRNA-20a on proliferation of pan- creatic carcinoma cells

目的观察慢病毒介导微小RNA（miRNA）-20a过表达对胰腺癌细胞增殖的影响，探讨胰腺癌基因治疗靶位。方法采用荧光原位杂交和实时定量逆转录-聚合酶链反应（RT—PCR）检测BxPC3、Panc-1和永生化的正常胰腺导管上皮细胞H6C7中miRNA-20a的表达。体外实验分析慢病毒介导miRNA-20a过表达对胰腺癌细胞株增殖能力的影响，流式细胞仪分析细胞周期分布。结果miRNA-20a在胰腺癌细胞中呈低表达（P〈0．01）；过表达miRNA-20a在体外可抑制胰腺癌细胞增殖（P〈0．01），生长曲线最大抑制效率分别是（59．21±0．31）％和（59．04±2．01）％；软琼脂克隆形成试验，增殖率分别下降到（45．21±1．72）％和（38．74±2．13）％。miRNA一20a使细胞周期阻滞于G0／G1期，分别为（62．3±4．5）％和（64．2±4．8）％（P〈0．05）。同时细胞周期蛋白D1（CyclinD1）表达水平下调，但其mRNA水平不变（P〉0．05）。结论miRNA-20a过表达能抑制胰腺癌细胞增殖，其机制可能是在转录后水平抑制CyclinD1蛋白的表达。

Objective To investigate the＇e^ffect of microRNA （miRNA）-20a overexpression medi- ated by lentivirus on pancreatic carcinoma cell proliferation, and to find new target spot of gene therapy for pancreatic carcinoma. Methods In situ hybridization and quantitative real-time reverse transcription-poly- merase chain reaction （RT-PCR） was used to evaluate the expression of miRNA-20a in two pancreatic car- cinoma cell lines （ BxPC3 and Panc-1 ） and immortal human pancreatic duct epithelial cell line H6C7. The effect of lentivirus-mediated overexpression of miRNA-20a on pancreatic carcinoma cell proliferation in vitrowas analyzed, and the cell cycle distribution was measured by using flow cytometry. Results The ex- pression level of miRNA-20a was downregulated in pancreatic carcinoma cells as compared with that in nor- mal pancreatic cells. Stable overexpression of miRNA-20a significantly inhibited proliferation of pancreatic carcinoma cells BxPC-3 and Panc-1 in vitro （P 〈0. 01 ）. The maximum inhibition rate of growth curve was （59. 21±0. 31 ）% and （59. 04 ±2. 01 ）% respectively. In soft agar colon formation test, the percentage of proliferation was decreased to （45.21 ± 1.7 ） % and （ 38.74 ± 2. 10 ） % respectively. Cell cycle distri- bution analysis showed there were great changes in miRNA-20a overexpressing clones. Many cells were blocked in the G0/GI phase, and the proportion of BxPC3 and Pane-1 cells was （62. 3 ± 4. 5） % and （64. 2±4. 8）% respectively （P 〈 0.05）. Furthermore, it was found that Cyclin D1 protein expression was downregulated by overexpression of miRNA-20a without changing the Cyelin D1 mRNA level. Conclu- sion Overexpression of miRNA-20a significantly inhibited proliferation of pancreatic carcinoma cells, and the probable mechanism was the negatively regulation of Cyelin D1 protein expression at the post-transcrip- tional level.