PEG10基因siRNA真核表达载体的构建及其对肝癌HepG2细胞凋亡的影响

目的:构建针对遗传印记基因PEG10的siRNA真核表达载体,体外观察其对肝癌HepG2细胞凋亡的影响. 方法:设计针对PEG10基因的3个siRNA靶序列,构建真核表达载体siRNA1,siRNA2和siRNA3,脂质体分别转染HepG2细胞后实时定量PCR,检测其对HepG2细胞PEG10基因表达的影响,MTT法、流式细胞仪和激光共聚焦检测转染siRNA后的HepG2细胞的生长活性及凋亡. 结果:成功构建了针对PEG10基因的3个特异性siRNA表达载体. 它们不同程度地抑制了HepG2细胞PEG10基因的表达,以siRNA2抑制效率最高,可达93.99%. HepG2细胞的生长也明显受到抑制(P＜0.05),大量HepG2细胞凋亡,siRNA2凋亡率最高,为66.91%. 结论:靶向PEG10基因的siRNA表达载体可抑制肝癌HepG2细胞的生长,诱导细胞凋亡,该作用与降低PEG10基因的表达有关.

Construction of siRNA eukaryotic expression vectors of PEG10 gene and their apoptosis-inducing effect on transfected HepG2 cells

AIM: To construct the siRNA eukaryotic expression vectors of PEG10 gene and to investigate their apoptosis-inducing effect on transfected HepG2 cells in vitro. METHODS: Three specific siRNAs of PEG10 were designed, and cloned into the psiRNA-hH1neo, which were transiently transfected into HepG2 via Lipofectamine 2000. Realtime quantitative PCR was used to measure the PEG10 mRNA expression in HepG2. The cell growth was assessed by MTT assay and the apoptosis was evaluated by flow cytometry and confocal microscopy. RESULTS: Three specific siRNA eukaryotic expression vectors (siRNA1, siRNA2, siRNA3) targeting PEG10 were successfully produced, which could inhibit the expression of PEG10 mRNA to varying degrees. The expression of PEG10 mRNA was remarkably inhibited at 48 h after transfection, and siRNA2 resulted in the highest inhibition rate (93.99%). HepG2 growth was also inhibited significantly (P<0.05), and cell apoptosis rate reached the highest after siRNA2transfection (66.91%). CONCLUSION: SiRNA eukaryotic expression vectors targeting PEG10 gene can induce HepG2 cell growth inhibition and apoptosis through down-regulating PEG10.