短发卡RNA介导的缺氧诱导因子-1α基因沉默对缺氧状态下骨肉瘤细胞增殖的影响

摘要

目的 观察缺氧环境对原代骨肉瘤细胞增殖的影响,以及用短发卡RNA (shRNA)沉默缺氧诱导因子(HIF)-1α基因后,缺氧状态下原代骨肉瘤细胞增殖的变化.方法 将原代骨肉瘤细胞分别置于缺氧环境(37℃、1％ O2、5％ CO2)或常氧环境(37℃、21％O2、5％ CO2)下培养,采用噻唑蓝(MTT)法检测细胞增殖,流式细胞仪检测细胞周期变化,Western blot检测HIF-1 α蛋白表达水平.其次,将HIF-1α基因及阴性对照基因(SCR)对应的shRNA由慢病毒载体介导转染原代骨肉瘤细胞,分别记为HIF-1 α/shRNA组和SCR/shRNA组,定量聚合酶链反应(qPCR)定量分析HIF-1αmRNA变化,Western blot检测转染后细胞HIF-1 α蛋白表达变化.最后,转染后的细胞再次缺氧培养,采用MTT法检测细胞增殖.结果 细胞实验显示,缺氧环境可在一定时间(24 h)内抑制原代骨肉瘤细胞增殖,但随着缺氧时间延长抑制作用减弱,与常氧组比较,缺氧细胞12 h细胞增殖率减少30.5％,24 h减少25.0％.同时HIF-1α表达升高,表明肿瘤细胞增殖与HIF-1α之间明显相关.转染shRNA后,与空白对照组及SCR/shRNA组比较,HIF-1 α/shRNA组的HIF-1α mRNA相对表达量明显降低(P =0.000),HIF-1α蛋白表达下降(P=0.000),缺氧培养的细胞增殖受到抑制.结论 shRNA介导的HIF-1 α基因沉默可以有效逆转缺氧环境中HIF-1 α对骨肉瘤细胞增殖的促进作用,从而可能干预肿瘤发育和生长的进程.%Objective To explore the effect of hypoxic condition on the proliferation of primary osteosarcoma cells,and the proliferation change of primary osteosarcoma cells under hypoxia after hypoxia inducible factor (HIF)-1 α gene silence with short hairpin RNA (shRNA).Methods Primary osteosarcoma cells were isolated from osteosarcoma tumor and cultured under hypoxic (37℃,1％ O2,5％ CO2) or nonnoxic (37℃,21％ O2,5％ CO2) condition separately.Firstly,the cell proliferation,cell cycle and the HIF-lα protein expression of the cells were detected by methyl thiazol tetrazolium (MTT) assay,flow cytometry and Western blotting separately.Secondly,the primary osteosarcoma cells were transfected by lentiviral vectors carrying HIF-1α or scramble (SCR) shRNA,which was respectively denoted as HIF-1 α/shRNA group or SCR/shRNA group.HIF-1 α mRNA expression in both groups was quantified using quantitative polymerase chain reaction (qPCR) and the HIF-1 α protein expression was detected by Western blotting.Finally,the transfected cells were cultured under hypoxic condition,and subsequently MTT assay was carried out for the evaluation of their proliferation.Results Hypoxia could inhibit the proliferation of primary osteosarcoma cells within 24 h,and this inhibition attenuated with the continually hypoxic condition.The expression of HIF-1 α increased under continually hypoxic condition,indicating that the osteosarcoma cell proliferation might be related to the HIF-1 α expression,and the cell proliferation rate of hypoxia cells decreased by 30.5％ in 12 h and 25.0％ in 24 h.As compared with SCR/shRNA group,the mRNA and protein expression levels of HIF-1α in HIF-1α/shRNA group significantly decreased (P =0.000),suggesting that the specific gene silence of HIF-1 α could inhibit the osteosarcoma cell proliferation.Conclusion shRNA-mediated HIF-1 α gene silence can effectively reverse the promoting effect of HIF-1 α on osteosarcoma cell proliferation under hypoxic condition,which may further interfere with the osteosarcoma development and growth.