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目的:探讨缺氧对人肝癌SMMC-7721细胞中缺氧诱导因子1α(hypoxia-inducible factor-1α,HIF-1α)和分化型胚胎软骨发育基因1(differentiated embryo-chondrocyte expressed gene 1,DEC1)表达的影响,并分析其机制,为肝癌的缺氧调节提供理论依据。方法:以肝癌SMMC-7721细胞为实验材料,应用二氯化钴(CoCl2)建立体外缺氧模型,对SMMC-7721细胞进行不同时间的缺氧培养(0、2、4、6、24和48h)。RT-PCR和蛋白质印迹法分别检测缺氧不同时相HIF-1α和DEC1mRNA及蛋白质的表达变化。HIF-1α和DEC1蛋白表达的相关性采用Pearson等级相关性分析。应用HIF-1α蛋白抑制剂YC-1对SMMC-7721细胞进行HIF-1α抑制培养,并设立缺氧对照组和常氧对照组,观察抑制HIF-1α对DEC1表达影响。结果:常氧培养时,肝癌细胞中HIF-1α和DEC1mRNA和蛋白表达明显高于肝细胞。随缺氧时间延长,HIF-1α蛋白、DEC1mRNA及DEC1蛋白表达均明显增加,F值分别为183.13、127.78及84.22,P值均<0.01;但HIF-1αmRNA表达无明显变化,F=0.960,P>0.05。其中HIF-1α蛋白、DEC1mRNA及DEC1蛋白均在缺氧4h达到高峰,相对表达量分别为1.183±0.063、2.182±0.234及0.839±0.051,与常氧对照组相比较,差异均有统计学意义,P值均<0.001。Pearson相关性分析结果显示,HIF-1α蛋白与DEC1蛋白表达呈高度正相关,r=0.826,P<0.05。YC-1抑制培养时,HIF-1α蛋白、DEC1mRNA及DEC1蛋白均较缺氧对照组明显降低,相对表达量分别为0.507±0.028、0.702±0.044及0.676±0.067,F值分别为428.12、730.24和371.69,P值均<0.01;但HIF-1αmRNA表达无明显变化,F=0.757,P>0.05。结论:干扰或抑制肝癌SMMC-7721细胞中HIF-1α蛋白的表达,能够降低缺氧诱导的DEC1高表达,为肝癌的缺氧调节和临床治疗提供理论依据。
Objective: To investigate the effects of hypoxia on the expression of hypoxia-inducible factor-1α (HIF-1α) and differentiated embryo-chondrocyte gene (DEC1) in human hepatoma SMMC-7721 cells , And analyze its mechanism to provide a theoretical basis for hypoxic regulation of liver cancer. Methods: The SMMC-7721 cells were used as experimental material to establish hypoxia model with cobalt chloride (CoCl 2) in vitro and hypoxic culture of SMMC-7721 cells at different time (0, 2, 4, 6, 24 and 48h ). RT-PCR and Western blotting were used to detect the expression of HIF-1α and DEC1mRNA and protein in different phases of hypoxia respectively. The correlation between HIF-1α and DEC1 protein expression was analyzed using Pearson's rank correlation analysis. HIF-1α was inhibited by HIF-1α protein inhibitor YC-1 and hypoxia control group and normoxia control group were established. The inhibitory effect of HIF-1α on DEC1 expression was observed. Results: Under normoxic culture, the expression of HIF-1α and DEC1 mRNA and protein in hepatocellular carcinoma cells was significantly higher than that in hepatocytes. The expression of HIF-1αprotein, DEC1mRNA and DEC1protein increased with time of hypoxia, the F values were 183.13, 127.78 and 84.22, respectively, P <0.01; however, the expression of HIF-1αmRNA had no significant change, F = 0.960, P > 0.05. The HIF-1αprotein, DEC1mRNA and DEC1protein peaked at4h after hypoxia, the relative expression levels were1.18 ± 0.063,2.182 ± 0.234and0.839 ± 0.051, respectively, which were significantly different from those in normoxia control group P <0.001. Pearson correlation analysis showed that there was a highly positive correlation between HIF-1α protein and DEC1 protein expression (r = 0.826, P <0.05). YC-1 inhibited the expression of HIF-1αprotein, DEC1mRNA and DEC1protein compared with hypoxia control group, the relative expression levels were0.507 ± 0.028,0.702 ± 0.044and0.676 ± 0.067, F values were428.12,730.24and 371.69, P values were all <0.01; however, there was no significant change in HIF-1αmRNA expression, F = 0.757, P> 0.05. CONCLUSION: Interference or inhibition of HIF-1α expression in hepatocellular carcinoma SMMC-7721 cells can reduce hypoxia-induced high expression of DEC1 and provide a theoretical basis for HCC hypoxia regulation and clinical treatment.