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目的构建针对Livin基因的短发夹RNA(shRNA)真核表达载体,探讨其对肝肿瘤HepG2细胞化疗敏感性的影响。方法设计并构建针对Livin基因的shRNA真核表达载体pSD11-U6/Neo/GFP/Livin,脂质体法转染肝肿瘤HepG2细胞。荧光定量PCR、Western blotting检测细胞Livin mRNA和蛋白的相对表达水平。顺铂(2.0 mg/L)处理后,MTT法检测细胞生长抑制率。结果测序分析证实针对Livin基因的shRNA真核表达载体构建成功,转染肝肿瘤HepG2细胞后可降低Livin mRNA和蛋白的相对表达量(P<0.05),顺铂对转染Livin表达载体的HepG2细胞抑制率明显高于未转染细胞(P<0.05)。结论成功构建Livin shRNA真核表达载体,且有效抑制HepG2细胞中Livin基因的表达,增强肝肿瘤细胞对化疗药物的敏感性。
Objective To construct an eukaryotic expression vector of short hairpin RNA (shRNA) against Livin gene and investigate its effect on chemosensitivity of HepG2 cells. Methods shRNA eukaryotic expression vector pSD11-U6 / Neo / GFP / Livin targeting Livin gene was designed and constructed and transfected into HepG2 cells by lipofectamine. Fluorescence quantitative PCR and Western blotting were used to detect the relative expression of Livin mRNA and protein. After treatment with cisplatin (2.0 mg / L), the cell growth inhibition rate was determined by MTT assay. Results Sequencing analysis confirmed that shRNA eukaryotic expression vector targeting Livin gene was constructed successfully, and the relative expression of Livin mRNA and protein was decreased after HepG2 cells were transfected with HepG2 cells (P <0.05). Cisplatin was transfected into HepG2 cells transfected with Livin expression vector The inhibition rate was significantly higher than that of untransfected cells (P <0.05). Conclusion The eukaryotic expression vector of Livin shRNA was successfully constructed and the expression of Livin gene in HepG2 cells was effectively inhibited. The sensitivity of chemosensitive drugs to liver cancer cells was enhanced.