摘要
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 mechanisms that microRNA221 （miR-221） promotes cancer development in hepatitis B virus （HBV）-related hepatocellular carcinoma （HCC） as well as HBx protein promotes HepG2 cells aberrant proliferation through up-regulation of miR-221. Methods Recombinant adenovirus with HBx gene （adv-HBx） was used to transfect HepG2 cells. The changes of miR-221 and estrogen receptor α （ERα） mRNA were validated by real-time fluorescence quantitative PCR, and the protein levels of ERα were quantified by Western blotting. Cell cycle was detected by flow cytometry. miR-221 mimic and miR-221 inhibitor were used to transfect HepG2 cells respectively. The changes of miR-221 and ERα mRNA in HBx-HepG2 cells were determined by real-time fluorescence quantitative PCR, and ERα protein levels were quantified by Western blotting. Results RT-PCR results confirmed that HBx was highly expressed in HepG2 cells after adv-HBx transfection. In 48 h after transfection, HBx protein enhanced the expression of miR-221 （495.84±61.16 vs 239.25±21.15, P＜0.05）, decreased ERα at protein level （0.24±0.01 vs 0.61±0.02, P＜0.05）, and promoted aberrant proliferation of HepG2 cells [（31.73±3.53）% vs （56.08±1.56）%, P=0.01]. Cell transfection and Western blotting results indilated that miR-221 decreased the expression of ERα protein （P〈0.05） while miR-221 inhibitor increased it （P〈0.05）, which suggested that ERα might be an important target of miR-221. Conclusion HBx may decrease the protective effects of ERα against HCC by up-regulation of miR-221, and then promote aberrant proliferation of hepatocarcinoma cells. miR-221 may be a potential target for hepatocarcinoma therapy.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2013年第20期2191-2194,共4页
Journal of Third Military Medical University
基金
国家自然科学基金(81071621
30973378
81101826
81272545)
国家临床重点专科建设项目[财社(2010)305号]
重庆市自然科学基金(2010BB5390)
重庆市卫生局课题(2010-2-090)~~
