siRNA沉默PPARα减弱羟喜树碱诱导的人结肠癌细胞凋亡

Silencing PPARα by siRNA Reduces Hydroxycamptothecin-induced Apoptosis in Human Colon Cancer Cells

背景：多药耐药一直是遏制提高结肠癌化疗有效性的瓶颈问题.目的：探讨沉默PPARα能否减弱羟喜树碱诱导的人结肠癌细胞凋亡,从而阐明PPARα在羟喜树碱耐药中的潜在作用.方法：构建针对PPARα基因的siRNA干扰载体,并转染人结肠癌SW1116细胞,经G418筛选出稳定转染的细胞,以RT-PCR和蛋白质印迹法验证干扰效果.经羟喜树碱干预后,以MTT法检测细胞增殖,Annexin V-FITC/PI检测细胞凋亡.以定量RT-PCR检测不同浓度miR-506前体转染SW1116细胞后对PPARα下游调控靶基因mRNA表达的影响.结果：siRNA干扰载体Ⅲ的沉默效果最佳,PPARα mRNA和蛋白表达分别下调了约94.1%±3.4%和70.8%±8.6%.稳定转染siRNA的SW1116细胞的半数抑制浓度（IC50）为（332±19）μg/L,亲本SW1116细胞为（152±12）μg/L.在相同浓度羟喜树碱的作用下,亲本SW1116细胞的凋亡率显著高于稳定转染siRNA的SW1116细胞（150μg/L：7.0%±2.5%对2.8%±1.6%;300μg,L：32.4%±7.1%对18.5%±2.7%）.miR-506前体下调HMGCS-2表达,上调FABP-2、CCND1和TGF-β1表达,其中上调CCND1的作用最为明显.结论：成功构建了,针对PPARα的siRNA干扰载体;沉默PPARα能增强SW1116细胞对羟喜树碱的抵抗性,可能与下调HMGCS-2表达并上调FABP-2、CCND1和TGF-β1表达有关.

Background： Multidrug resistance is a significant impediment to the success of chemotherapy in colon cancer. Aims： To appraise whether PPARα silencing reduces hydroxycamptothecin-induced apoptosis in human colon cancer cells so as to elucidate the potential role of PPARoL in hydroxycamptothecin-related resistance. Methods： siRNA vectors for silencing PPARct were constructed and transfected into human colon cancer SW1116 cells. The transfected SW1116 cells were cultured in G418-selective medium for screening. The silencing effect was verified by RT-PCR and Western blotting. After treated with hydroxycamptothecin, cell proliferation was assayed by MIT method, and Annexin V-FITC/PI was used for measuring apoptosis rate. Quantitative RT-PCR was used to determine the mRNA expressions of HMGCS-2, FABP-2, CCND1 and TGF-131, the downstream regulating genes of PPARα, when different concentrations of miR-506 precursor were transfected into SW1116 cells. Results： siRNA construction Ⅲ had the best silencing effect, which down- regulated the expression of PPARα mRNA by 94.1%＋3.4% and PPARα protein by 70.8%＋8.6%. IC50 was （332±19） μg/L for siRNA-transfected SWlll6 cells and （152±12）μg/L for SW1116 cells. At the same concentration of hydroxycamptothecin, the apoptosis rate of SW1116 cells was significantly higher than that of siRNA-transfected SW1116 cells （150μg/L：7.0%±2.5%对2.8%±1.6%;300μg,L：32.4%±7.1%对18.5%±2.7%）. miR-506 precursor down- regulated the expression of HMGCS-2 and up-regulated the expressions of FABP-2, CCND1 and TGF-β1, the most significantly up-regulated was CCNDI. Conclusions： siRNA for PPARα is successfully constructed. Silencing PPARα can increase the resistant ability of SW1116 cells to hydroxycamptothecin, which may be related to the down-regulation of expression of HMGCS-2 and up-regulation of expressions of FABP-2, CCND1 and TGF-β1.