HIF-1α基因沉默对大鼠肝癌CBRH-7919细胞p27和Ki67表达的影响

Effects of HIF-1αsilencing on expression of p27 and Ki67 in rat hepatoma cell line CBRH-7919

目的:探讨沉默缺氧诱导因子1α(hypoxia-inducible factor 1α,HIF-1α)基因对大鼠肝癌CBRH-7919细胞在缺氧状态下p27和Ki67表达的影响。方法:选用大鼠肝癌细胞株CBRH-7919作为研究对象,利用氯化钻(CoCl2)模拟缺氧状态。构建HIF-1αsiRNA特异性载体,利用小分子RNA干扰技术沉默肝癌细胞HIF-1α基因。应用real-time RT-PCR和Western blotting方法分别检测肝癌细胞HIF-1αmRNA和蛋白的表达变化,用Western blotting方法检测肝癌细胞p27和Ki67蛋白的表达变化。应用流式细胞术检测细胞周期的变化。结果:在缺氧条件下,肝癌细胞HIF-1αmRNA及蛋白表达显著增多(P<0.05)。HIF-1α基因被沉默后,HIF-1αmR-NA和蛋白表达以及Ki67蛋白表达量明显减少(P<0.05),p27蛋白表达量显著增多(P<0.05)。转染组G0/G1期的肝癌细胞数量明显多于对照组,S期细胞显著减少(P<0.05)。结论:缺氧可以诱导肝癌细胞HIF-1α的表达;特异性siRNA沉默HIF-1α,可能通过促进p27的表达和抑制Ki67的表达,在一定程度上抑制了肝癌细胞的增殖。

AIM： To study the effect of hypoxia -inducible factor 1 α （HIF- 1α） silencing on the expression of p27 and Ki67 in rat hepatoma cell line CBRH -7919 under hypoxia. METHODS： Hypoxic condition was induced by CoCl2 and the expression of HIF- 1α was silenced by small interference RNA. HIF-1α- specific RNAi lentiviral vector was constructed. Real - time RT - PCR and Western blotting were used to detect the expression of HIF - 1 et at mRNA and protein levels in CBRH -7919 cells under hypoxia. The expression of p27 and Ki67 was observed by Western blotting after HIF -1α silencing was performed. The cell cycle of hepatoma cells was detected by flow cytometry. RESULTS ： Under hypexic condition, the expression of HIF- 1α at mRNA and protein levels in CBRH-7919 cells increased significantly （P 〈 0. 05）. HIF - 1α silencing significantly reduced the expression of Ki67 but increased the expression of p27 （P 〈 0. 05 ） in CBRH - 7919 cells. In transfected cells, the number of cells in G0/G, phase was much higher and that in S phase was much lower than those in the control cells. CONCLUSION： Hypexia induces the expression of HIF - 1α. HIF -1α silencing can regulate the proliferation of hepatoma ceils through reducing the expression of Ki67 and increasing the expression of p27.