【摘要】 目的观察应用短发夹RNA(shRNA)沉默缺氧诱导因子(HIF)-1α对肝癌细胞化疗敏感性的影响并探讨其相关机理。方法脂质体包裹HIF-1αshRNA表达载体(pshRNA-HIF-1α)转染到肝癌细胞HepG2细胞中,G418筛选,建立稳定的HIF-1α沉默的细胞系,并以转染空白质粒(pHK)作对照。逆转录聚合酶链反应(RT-PCR)检测沉默HIF-1α后HIF-1αmRNA和多药耐药基因1(mdr1)mRNA变化;免疫印迹(Western blot)检测细胞HIF-1α及P-糖蛋白(P-gp)的变化。CoCl2模拟缺氧环境下,MTT和流式细胞术检测不同剂量的化疗药物(阿霉素)在HIF-1α沉默后对细胞生长抑制率和凋亡率的影响。结果pshRNA-HIF-1α转染细胞后HIF-1α干扰率在mRNA水平和蛋白水平分别为82.18%和75.51%。沉默HIF-1α后细胞mdr1 mRNA和P-gp表达显著降低(P<0.05),生长抑制率和凋亡率明显增高(P<0.05)。结论shRNA沉默HepG2细胞HIF-1α可以通过下调mdr1 mRNA和P-gp表达逆转肝癌化疗耐药,增强肝癌细胞对化疗的敏感性;可通过沉默HIF-1α作为肝癌基因治疗的一种新途径。更多还原

【Abstract】 Objective To observe the effect of disruption of hypoxia inducible factor-1α(HIF-1α) pathway by small hairpin RNA(shRNA) on chemosensitivity of human hepatocellular carcinoma(HCC) cells and to reveal the correlative mechanisms.Methods Plasmid of pshRNA-HIF-1α was transfected into HepG2 cells by lipofectamine.HepG2/pshRNA-HIF-1α(HepG2/pshRNA) cell lines were obtained by selection of HepG2 cells in G418.Meanwhile,plasmid of empty vector(pHK) was transfected as a control(HepG2/pHK).The mRNA and protein expression levels of HIF-1α and mdr1 were investigated by RT-PCR and Western blot respectively.Using CoCl2 to simulate the hypoxia condition,growth inhibition and apoptosis rates of HepG2 cells under different dosages of chemotherapeutic agents(adriamycin) were measured by MTT assay and flow cytometry(FCM).Results Compared with HepG2/pHK cells,the mRNA and protein expression levels of HIF-1α and mdr1 were obviously down-regulated in HepG2/pshRNA cells.At the same time,the proliferation inhibition and apoptosis rates were evidently increased after transfection with pshRNA-HIF-1α(P<0.05),which decreased the expression of HIF-1α to 82.18% at mRNA level and 75.51% at protein level.There was no significant effect of transfection pHK(P>0.05).Conclusion These data demonstrates that HIF-1α interference by shRNA increased the sensitivity of HCC chemotherapy and the reversal of multidrug resistance,which may be done by down-regulating the transcription of mdr1 and the translation of P-gp.Blocking HIF-1α in HCC cells may offer an new avenue for gene therapy.更多还原