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目的探讨microRNA221(miR-221)在乙型肝炎病毒(hepatitis B virus,HBV)感染相关性肝细胞癌(hepatocellular carcinoma,HCC)中的促癌功能以及HBV基因编码的X蛋白(HBx)诱导miR-221上调促进HepG2细胞异常增殖的分子机制。方法重组HBx腺病毒(Ad-HBx)感染人肝癌HepG2细胞,荧光定量PCR检测HepG2-HBx细胞中miR-221和ERα mRNA表达的变化;流式细胞术检测细胞周期,Western blot检测雌激素受体α(estrogen receptorα,ERα)蛋白表达水平变化,分别用miR-221 mimic和miR-221 inhibitor转染HepG2细胞后,荧光定量PCR检测HepG2-HBx细胞中miR-221和ERαmRNA表达变化;Western blot检测ERα蛋白水平表达变化。结果 RT-PCR实验证实,adv-HBx感染HepG2细胞后,HBx在HepG2细胞中高效表达;感染48 h后,HBx蛋白可显著上调miR-221[(495.84±61.16)vs(239.25±21.15),P<0.05]并抑制ERα蛋白[(0.24±0.01)vs(0.61±0.02),P<0.05]的表达水平,同时促进HepG2细胞异常增殖[(31.73±3.53)%vs(56.08±1.56)%,P=0.01]。miRNA转染实验及Western blot证实:miR-221抑制ERα蛋白的表达(P<0.05),miR-221抑制剂促进ERα蛋白的表达(P<0.05)。结论 HBx可能通过上调miR-221进而下调ERα对肝癌的保护性效应而促进肝癌细胞异常增殖,靶向miR-221的策略具有抑制肝癌细胞增殖的治疗潜能。
Objective To investigate the role of miR-221 in promoting tumor-promoting function in hepatitis B virus (HBV) -related hepatocellular carcinoma (HCC) and the role of miR-221 in inducing miR- 221 Up-regulates the Molecular Mechanism of Abnormal Proliferation in HepG2 Cells. Methods HepG2 cells were infected with recombinant HBx adenovirus (Ad-HBx) and the expression of miR-221 and ERα mRNA in HepG2-HBx cells was detected by real-time PCR. Cell cycle was detected by flow cytometry and estrogen receptor 221 mRNA and miR-221 inhibitor were transfected into HepG2 cells respectively. The expression of miR-221 and ERαmRNA in HepG2-HBx cells was detected by real-time PCR. Western blot was used to detect the expression of ERα Changes in protein expression. Results RT-PCR showed that HBx was highly expressed in HepG2 cells after infection with HepG2 cells. The expression of HBx protein was significantly up-regulated in HepG2 cells 48 h after infection (P <0.001) <0.05] and inhibited the expression of ERα protein [(0.24 ± 0.01) vs (0.61 ± 0.02), P <0.05], and promoted the abnormal proliferation of HepG2 cells [(31.73 ± 3.53)% vs (56.08 ± 1.56)% = 0.01]. miRNA transfection experiments and Western blot confirmed that miR-221 inhibited the expression of ERα protein (P <0.05), and miR-221 inhibitor promoted the expression of ERα protein (P <0.05). Conclusion HBx may promote the abnormal proliferation of hepatoma cells by upregulating the expression of miR-221 and down-regulating the protective effect of ERα on hepatocellular carcinoma. The targeting strategy of miR-221 has the potential to inhibit the proliferation of hepatocellular carcinoma cells.