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目的探讨micro RNA-10b(mi R-10b)及其靶基因同源异性盒基因HOXD10在肝癌细胞中的表达及转染mi R-10b对肝癌细胞侵袭性能力的影响。方法分析HOXD10基因在3种肝癌细胞株Huh7、Hep G2、SMMC-7721中的基础表达量,筛选出HOXD10基础表达量高的Hep G2作为后续研究对象。设计合成外源性mi R-10b序列,用脂质体瞬时转染肝癌细胞株,设置mi R-10b转染组、无关序列转染组(阴性对照组)和未处理组(对照组)。用RT-PCR及Western blot检测肝癌细胞转染mi R-10b后HOXD10基因及蛋白表达量的变化,细胞侵袭实验研究转染mi R-10b对肝癌细胞侵袭能力。结果在基因水平和Western blot分析蛋白水平HOXD10基因表达量2-ΔΔCT分别是(1.24±0.23)、(1.00±0.02),P>0.05差异无统计学意义。在蛋白分析Hep G2细胞灰度计算值是值分别是653.492和1 968.742,蛋白水平表达降低。HOXD10基因在肝癌细胞株Hep G2的表达量显著高于Huh7和SMMC-7721;mi R-10b成功转染Hep G2后,HOXD10基因表达水平与对照组比较无明显变化,但HOXD10蛋白表达量与对照组比较下降及侵袭性也增强。结论 mi R-10b可能通过抑制靶基因HOXD10表达发挥作用,促进肝癌细胞的侵袭。
Objective To investigate the expression of microRNA-10b (miR-10b) and its target gene homologous box gene HOXD10 in hepatoma cells and the effect of transfected mi R-10b on the invasiveness of hepatoma cells. METHODS: The basic expression level of HOXD10 gene in three hepatocellular carcinoma cell lines Huh7, Hep G2 and SMMC-7721 was analyzed. Hep G2 with high basic expression of HOXD10 was screened as a follow-up study. The exogenous mi R-10b sequence was designed and synthesized. The liver cancer cell line was transiently transfected with liposome. The mi R-10b transfection group, the irrelevant sequence transfection group (negative control group) and the untreated group (control group) were set. RT-PCR and Western blot were used to detect the changes of HOXD10 gene and protein expression in hepatoma cells transfected with miR-10b. Cell invasion assays were used to study the invasion ability of mi R-10b transfected hepatoma cells. Results The 2-ΔΔCT of HOXD10 gene expression at gene level and Western blot analysis were (1.24±0.23) and (1.00±0.02), respectively. There was no significant difference between P>0.05. The calculated values for gray scale values in protein analysis of Hep G2 cells were 653.492 and 1 968.742, respectively, and protein level expression was decreased. The expression level of HOXD10 gene in Hep G2 was significantly higher than that of Huh7 and SMMC-7721. After miR-10b was successfully transfected into Hep G2, the level of HOXD10 gene expression was not significantly different from that of the control group, but HOXD10 protein expression level was correlated with that of the control group. The group’s decline and invasiveness also increased. Conclusion mi R-10b may play a role in inhibiting the expression of target gene HOXD10 and promote the invasion of hepatoma cells.