上调微小RNA-148a对胰腺癌AsPC-1细胞抑癌基因前脑啡肽原、p16和Ras相关区域家族1A基因表达的影响

The effects of up - regulation of microRNA - 148a on the expression of anti - oncogene preproenkephalin, p16 and ras association domain family 1A in pancreatic carcinoma AsPC -1 cells

目的观察上调微小RNA-148a（miR-148a）表达对胰腺癌AsPC-1细胞抑癌基因前脑啡肽原（ppENK）、p16和Ras相关区域家族1A基因（RASSF1A）甲基化、蛋白表达及细胞增殖的影响。方法利用Lipofectamine？2000转染miR-148a模拟物（miR-148a mimics）至胰腺癌AsPC-1细胞。实验细胞分为3组：空白对照组（转染脂质体）、阴性对照组（转染阴性对照序列）、实验组（转染miR-148a mimics）。转染后72 h，采用实时定量聚合酶链反应（Real-time PCR）检测细胞中miR-148a表达水平；甲基化特异性聚合酶链反应（MSP）检测ppENK、p16和RASSF1A基因启动子甲基化；Western blot法检测DNA甲基转移酶1（DNMT1）及ppENK、p16和RASSF1A蛋白表达水平；噻唑蓝（MTT）法检测细胞体外增殖活力。结果实验组miR-148a的表达量显著高于空白对照组及阴性对照组（4.63±1.32比1.00±0.30比0.93±0.37，P＝0.002），而DNMT1蛋白表达量则显著降低（1.00±0.00比1.04±0.19比0.27±0.15，P＝0.001）。空白对照组和阴性对照组ppENK、p16和RASSF1A基因甲基化阳性，而实验组ppENK和p16基因甲基化阴性，RASSF1A基因部分甲基化。实验组ppENK、p16和RASSF1A蛋白表达量均显著高于两个对照组（ppENK：2.06±0.32比1.00±0.00比1.01±0.21，P＝0.002；p16：2.02±0.12比1.00±0.00比0.96±0.12，P＝0.000；RASSF1A：1.95±0.37比1.00±0.00比1.09±0.12，P＝0.002）。实验组细胞体外增殖活力显著减慢（P＝0.026）。结论上调胰腺癌AsPC-1细胞miR-148a的表达后，抑癌基因ppENK、p16和RASSF1A发生去甲基化，蛋白质重新表达，并抑制癌细胞增殖，其作用机制与miR-148a靶向抑制DNMT1表达有关。

Objective To investigate the effect of up-regulation of microRNA （miRNA, miR）-148a on the methylation and protein expression of anti-oncogene preproenkephalin （ppENK）, p16, ras association domain family 1A （RASSF1A） and the proliferation of pancreatic cancer AsPC-1 cells.MethodsMiR-148a mimics were transfected into AsPC-1 cells mediated by Lipofectamine 2000. AsPC-1 cells were devided into three groups, blank group （transfected Lipofectamine）, mimincs-NC group （transfected negative mimics） and mimincs-148a group （transfected miR-148a mimics）. At 72 h after transfection, the expression of miR-148a was analyzed by real-time quantitative polymerase chain reaction （Real-time PCR）. The CpG island methylation status of anti-oncogene ppENK, p16, RASSF1A were measured by methylation specific polymerease chain reaction （MSP）. The expression of DNA methy transferas 1 （DNMT1）, ppENK, p16 and RASSF1A protein were detected with Western blotting. The cell proliferation activity was examined by methyl thiazol tetrazolium （MTT） assay.ResultsThe expression of miR-148a in mimincs-148a group was significantly higher than those in blank group and mimincs-NC group （4.63±1.32 vs. 1.00±0.30 vs. 0.93±0.37, P=0.002）, while the expression of DNMT1 protein in mimincs-148a group was significantly lower than those in blank group and mimincs-NC group （1.00±0.00 vs. 1.04±0.19 vs. 0.27±0.15, P=0.001）. ppENK, p16 and RASSF1A genes were positive methylation in blank group and mimincs-NC group, while ppENK and p16 genes were negative methylation, and RASSF1A gene was partial methylation in mimincs-148a group. The expression of ppENK, p16 and RASSF1A protein in mimics-148a group were significantly higher than those in blank group and mimics-NC group （ppENK： 2.06±0.32 vs. 1.00±0.00 vs. 1.01±0.21, P=0.002; p16： 2.02±0.12 vs. 1.00±0.00 vs. 0.96±0.12, P=0.000; RASSF1A： 1.95±0.37 vs. 1.00±0.00 vs. 1.09±0.12, P=0.002）. The cell proliferation activities in mimincs-148a group were significantly slower than those in in blank group and mimincs-NC group （P=0.026）.ConclusionUp-regulation of miR-148a could lead to demethylation of ppENK, p16 and RASSF1A gene promoter CpG island and up-regulate its protein expression, and inhibit cell proliferation in pancreatic cancer AsPC-1 cells. The mechanism may be related to the inhibition of DNMT1 expression induced by miR-148a.