摘要
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.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2012年第9期1771-1774,共4页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目(30972703、81171653)
江苏省卫生厅医学科技发展基金资助项目(17200932、P200933)
六大人才高峰第六批资金资助项目(BRA2010037)
常州市卫生局青年人才科技项目(QN201103)
关键词
胰腺癌
微小RNA
增殖
Pancreatic carcinoma
MicroRNA
Proliferation