siRNA-mediated Bcl-2 and Bcl-xl gene silencing sensitizes human hepatoblastoma cells to chemotherapeutic drugs
siRNA-mediated Bcl-2 and Bcl-xl gene silencing sensitizes human hepatoblastoma cells to chemotherapeutic drugs
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DOI：
10.1111/j.1440-1681.2007.04593.x
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发表时间：
2007-05-01
影响因子：
2.9
通讯作者：
Liao, Duan-Fang
中科院分区：
文献类型：
--
作者：
Lei, Xiao-Yong;Zhong, Miao;Liao, Duan-Fang
1.目的：1.研究Bcl-2和Bcl-xl siRNA表达载体转染HepG 2细胞后化疗药物敏感性的变化.构建Bcl-2和Bcl-xl siRNA及阴性siRNA表达载体，并稳定转染HepG 2细胞。采用逆转录聚合酶链反应（RT-PCR）检测目的基因的表达，免疫印迹法和免疫荧光法检测Bcl-2、Bcl-xl、Bax和caspase-3蛋白的表达。采用MTT法和流式细胞仪检测细胞对5-氟尿嘧啶（5-FU）和10-羟基喜树碱（HCPT）的敏感性. Bcl-2 siRNA、Bcl-xl siRNA和Bcl-2/Bcl-xl siRNA转染的细胞中Bcl-2和Bcl-xl基因表达和相应蛋白水平与阴性siRNA转染或未处理的细胞相比降低。Bax蛋白水平保持不变，但caspase-3水平增加时，Bcl-2和Bcl-xl蛋白水平降低。MTT结果显示Bcl-2和Bcl-xl转染细胞对5-FU和HCPT的敏感性增加。流式细胞术显示，与阴性siRNA或未处理的细胞相比，Bcl-2/Bcl-xl siRNA共转染的细胞以及Bcl-xl siRNA和Bcl-2 siRNA转染的细胞中亚G1细胞群增加。后一种趋势在5-FU或HCPT存在下进一步加强。因此，Bcl-2和Bcl-xl siRNA介导的基因沉默，与化疗相结合，可能是一个潜在的治疗策略，对人类肝母细胞瘤。
1. The aim of the present study was to investigate the changes in chemotherapeutic drug sensitivity of HepG2 cells transfected with Bcl-2 and Bcl-xl siRNA expression vectors.2. Bcl-2 and Bcl-xl siRNA and negative siRNA expression vectors were constructed and stably transfected into HepG2 cells. Reverse transcriptase-polymerase chain reaction was used to detect the target gene expression, and the Bcl-2, Bcl-xl, Bax and caspase-3 protein levels were measured using western blots and immunofluorescence. The sensitivity of the cells to the chemotherapeutic drugs 5-fluorouracil (5-FU) and 10-hydroxycamptothecin (HCPT) was analysed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) and flow cytometry.3. The Bcl-2 and Bcl-xl gene expression and corresponding protein levels in Bcl-2 siRNA, Bcl-xl siRNA and Bcl-2/Bcl-xl siRNA transfected cells were reduced compared with negative siRNA transfected or untreated cells. The Bax protein level remained unaltered but the caspase-3 level was enhanced when Bcl-2 and Bcl-xl protein levels were reduced. The MTT results demonstrated that Bcl-2 and Bcl-xl transfected cells exhibited increased sensitivity to 5-FU or HCPT. Flow cytometry demonstrated that the sub G1 cell population increased in Bcl-2/Bcl-xl siRNA co-transfected and Bcl-xl siRNA and Bcl-2 siRNA transfected cells when compared with negative siRNA or untreated cells. The latter trend was strengthened further in the presence of 5-FU or HCPT.4. Thus, Bcl-2 and Bcl-xl siRNA-mediated gene silencing, in combination with chemotherapy, may be a potential therapeutic strategy against human hepatoblastoma.