癌,肝细胞%RNA%肝炎病毒,乙型%突变%细胞增殖
癌,肝細胞%RNA%肝炎病毒,乙型%突變%細胞增殖
암,간세포%RNA%간염병독,을형%돌변%세포증식
Carcinoma,hepatocellular%RNA%Hepatitis B virus%Mutation%Cell proliferation
目的 研究微小RNA (miRNA)在HBx缺失突变体致人肝细胞异常增殖中的作用及其相关机制. 方法 利用miRNA芯片技术检测稳定表达HBx-d382及HBx的L02(即分别含HBx基因缺失突变体HBx-d382及野生型HBx基因)细胞系中miRNA的表达,实时荧光定量PCR对上述miRNA进行验证.选取在L02/HBx-d382和L02/HBx细胞中均表达明显下调的两个miRNA:miR-338-3P、miR-551b进行功能研究,分为实验组(转染miRNA模拟物组)、阴性对照组(NC,转染miRNA阴性对照)及脂质体组(lipo,单加转染试剂),通过脂质体转染到细胞中,四甲基偶氮唑盐法检测细胞存活率,流式细胞仪检测细胞周期,实时荧光定量PCR和Wstern blot检测细胞周期蛋白D1、细胞周期蛋白G1和E2F转录因子的改变.采用SPSS 16.0统计软件,数据均进行了正态检验并符合正态性,组间均数比较采用成组t检验. 结果 (1)与L02/pcDNA3.0细胞比较,L02/HBx-d382细胞有6个miRNA表达上调,5个miRNA表达下调; L02/HBx细胞有4个miRNA表达上调,12个miRNA表达下调.实时荧光定量PCR验证的结果与芯片相一致.(2)转染了miR-338-31p-模拟物和miR-551b-模拟物后,L02/HBx-d382及L02/HBx细胞增殖均受抑制,细胞周期均阻滞在G1期,细胞增殖能力减弱.L02/HBx-d382细胞中,细胞增殖能力:lipo组、NC组、miR-338-3p组和miR-551b组分别为90.0％±1.3％、88.0％±1.6％、56.0％±6.1％和62.0％±6.4％,miR-338-3p组与:lipo组、NC组比较,t值分别为10.402、9.133 ;miR-551b组和与lipo组、NC组比较,t值分别为8.763、7.403,P值均＜0.01.L02/HBx细胞中,细胞增殖能力:lipo组、NC组、miR-338-3p组和miR-551b组分别为91.0％±1.7％、89.0％±2.1％、60.0％±7.7％和66.0％±9.3％,miR-338-3p组与lipo组、NC组比较,t值分别为9.105、8.074;miR-551b组与lipo组、NC组比较,t值分别为7.673、7.52,P值均＜0.01.实时荧光定量PCR检测结果显示细胞周期蛋白D1、细胞周期蛋白G1和E2F转录因子的mRNA水平均无明显改变,但Western blot法发现上述基因的蛋白表达均明显下调,差异有统计学意义. 结论 ①HBx及其缺失突变体能影响L02细胞的miRNA表达谱;②在L02/HBx-d382细胞中下调更明显的miR-338-3p和miR-551b可能通过调控细胞周期蛋白D1、细胞周期蛋白G1和E2F转录因子的表达而调节了细胞的生长.
目的 研究微小RNA (miRNA)在HBx缺失突變體緻人肝細胞異常增殖中的作用及其相關機製. 方法 利用miRNA芯片技術檢測穩定錶達HBx-d382及HBx的L02(即分彆含HBx基因缺失突變體HBx-d382及野生型HBx基因)細胞繫中miRNA的錶達,實時熒光定量PCR對上述miRNA進行驗證.選取在L02/HBx-d382和L02/HBx細胞中均錶達明顯下調的兩箇miRNA:miR-338-3P、miR-551b進行功能研究,分為實驗組(轉染miRNA模擬物組)、陰性對照組(NC,轉染miRNA陰性對照)及脂質體組(lipo,單加轉染試劑),通過脂質體轉染到細胞中,四甲基偶氮唑鹽法檢測細胞存活率,流式細胞儀檢測細胞週期,實時熒光定量PCR和Wstern blot檢測細胞週期蛋白D1、細胞週期蛋白G1和E2F轉錄因子的改變.採用SPSS 16.0統計軟件,數據均進行瞭正態檢驗併符閤正態性,組間均數比較採用成組t檢驗. 結果 (1)與L02/pcDNA3.0細胞比較,L02/HBx-d382細胞有6箇miRNA錶達上調,5箇miRNA錶達下調; L02/HBx細胞有4箇miRNA錶達上調,12箇miRNA錶達下調.實時熒光定量PCR驗證的結果與芯片相一緻.(2)轉染瞭miR-338-31p-模擬物和miR-551b-模擬物後,L02/HBx-d382及L02/HBx細胞增殖均受抑製,細胞週期均阻滯在G1期,細胞增殖能力減弱.L02/HBx-d382細胞中,細胞增殖能力:lipo組、NC組、miR-338-3p組和miR-551b組分彆為90.0％±1.3％、88.0％±1.6％、56.0％±6.1％和62.0％±6.4％,miR-338-3p組與:lipo組、NC組比較,t值分彆為10.402、9.133 ;miR-551b組和與lipo組、NC組比較,t值分彆為8.763、7.403,P值均＜0.01.L02/HBx細胞中,細胞增殖能力:lipo組、NC組、miR-338-3p組和miR-551b組分彆為91.0％±1.7％、89.0％±2.1％、60.0％±7.7％和66.0％±9.3％,miR-338-3p組與lipo組、NC組比較,t值分彆為9.105、8.074;miR-551b組與lipo組、NC組比較,t值分彆為7.673、7.52,P值均＜0.01.實時熒光定量PCR檢測結果顯示細胞週期蛋白D1、細胞週期蛋白G1和E2F轉錄因子的mRNA水平均無明顯改變,但Western blot法髮現上述基因的蛋白錶達均明顯下調,差異有統計學意義. 結論 ①HBx及其缺失突變體能影響L02細胞的miRNA錶達譜;②在L02/HBx-d382細胞中下調更明顯的miR-338-3p和miR-551b可能通過調控細胞週期蛋白D1、細胞週期蛋白G1和E2F轉錄因子的錶達而調節瞭細胞的生長.
목적 연구미소RNA (miRNA)재HBx결실돌변체치인간세포이상증식중적작용급기상관궤제. 방법 이용miRNA심편기술검측은정표체HBx-d382급HBx적L02(즉분별함HBx기인결실돌변체HBx-d382급야생형HBx기인)세포계중miRNA적표체,실시형광정량PCR대상술miRNA진행험증.선취재L02/HBx-d382화L02/HBx세포중균표체명현하조적량개miRNA:miR-338-3P、miR-551b진행공능연구,분위실험조(전염miRNA모의물조)、음성대조조(NC,전염miRNA음성대조)급지질체조(lipo,단가전염시제),통과지질체전염도세포중,사갑기우담서염법검측세포존활솔,류식세포의검측세포주기,실시형광정량PCR화Wstern blot검측세포주기단백D1、세포주기단백G1화E2F전록인자적개변.채용SPSS 16.0통계연건,수거균진행료정태검험병부합정태성,조간균수비교채용성조t검험. 결과 (1)여L02/pcDNA3.0세포비교,L02/HBx-d382세포유6개miRNA표체상조,5개miRNA표체하조; L02/HBx세포유4개miRNA표체상조,12개miRNA표체하조.실시형광정량PCR험증적결과여심편상일치.(2)전염료miR-338-31p-모의물화miR-551b-모의물후,L02/HBx-d382급L02/HBx세포증식균수억제,세포주기균조체재G1기,세포증식능력감약.L02/HBx-d382세포중,세포증식능력:lipo조、NC조、miR-338-3p조화miR-551b조분별위90.0％±1.3％、88.0％±1.6％、56.0％±6.1％화62.0％±6.4％,miR-338-3p조여:lipo조、NC조비교,t치분별위10.402、9.133 ;miR-551b조화여lipo조、NC조비교,t치분별위8.763、7.403,P치균＜0.01.L02/HBx세포중,세포증식능력:lipo조、NC조、miR-338-3p조화miR-551b조분별위91.0％±1.7％、89.0％±2.1％、60.0％±7.7％화66.0％±9.3％,miR-338-3p조여lipo조、NC조비교,t치분별위9.105、8.074;miR-551b조여lipo조、NC조비교,t치분별위7.673、7.52,P치균＜0.01.실시형광정량PCR검측결과현시세포주기단백D1、세포주기단백G1화E2F전록인자적mRNA수평균무명현개변,단Western blot법발현상술기인적단백표체균명현하조,차이유통계학의의. 결론 ①HBx급기결실돌변체능영향L02세포적miRNA표체보;②재L02/HBx-d382세포중하조경명현적miR-338-3p화miR-551b가능통과조공세포주기단백D1、세포주기단백G1화E2F전록인자적표체이조절료세포적생장.
Objective To study the effect of micro (mi)RNA on cellular proliferation induced by hepatitis B x protein,HBx,in human liver cells and to investigate the underlying molecular mechanism of this cancer-related effect.Methods The human L02 hepatocyte cell line was stably transfected with HBx (L02/HBx) or an HBx mutant (L02/HBx-d382) that induces higher levels of cellular proliferation.The differential miRNA expression profiles were determined by microarray analysis and confirmed by realtime PCR.Two miRNAs,miR-338-3p and miR-551b,that were found to be significantly down-regulated in the L02/HBx-d382 cells were selected for further study and transfected individually into cells using the lipofectamine procedure.The cell survival rate was analyzed by MTT assay,and cell cycles were assessed by flow cytometry.Expressions of cyclinD1,cyclinG1,and E2F1 were assessed by real-time PCR and Western blotting.Results Compared with the microarray miRNA profile of L02/pcDNA3.0 cells,six miRNAs were up-regulated and five miRNAs were down-regulated in the L02/HBx-d382 cells,while four miRNAs were upregulated and 12 were down-regulated in the L02/HBx cells.The microarray results were consistent with realtime PCR results.Transfection of miR-338-3p and miR-551b significantly inhibited the cell survival rates (P＜0.001) and induced G0/G1 phase cycle arrest According to MTT results:for L02/HBx-d382 cells,compared with lipofectamine or non-transfected (NC) controls,the t value of miR-338-3p was 10.402,9.133 and the t value of miR-551b was 8.763,7.403; for L02/HBx cells,compared with lipofectamine or NC controls,the t value of miR-338-3p was 9.105,8.074 and the t value of miR-551b was 7.673,7.52.According to flow cytometry results:for L02/HBx-d382 cells,compared with lipofectamine or NC controls,the t value of miR-338-3p was 12.173,11.107and the t value of miR-55 1b was 15.364,13.377; for L02/HBx cells,compared with lipofectamine or NC controls,the t value of miR-338-3p was 15.416,13.378,and the t value of miR-551 b was 13.276,13.109.The protein levels of cyclinD1,cyclinG1,and E2F1 were significantly reduced by both miR-338-3p and miR-551b (P＜0.001).For L02/HBx-d382 cells,compared with lipofectamine or NC controls:E2F1 had t=11.132,10.031 and 12.017,10.973,respectively; cyclinD1 had t=15.654,15.013 and 15.447,14.733,respectively; cyclinG1 had t=8.017,7.661 and 7.402,7A17,respectively.For L02/HBx cells,compared with lipofectamine or NC controls:E2F1 had t=14.244,13.331 and 15,022,14.468,respectively; cyclinD1 had t=8.695,8.137 and 7.877,7.503,respectively;cyclinG1 had t=7.73,7.471 and 7.596,7.41,respectively.In contrast,the mRNA levels for E2F1,cyclinD1,and cylcinG1 showed no significant differences between the miRNA transfected cells and controls.Conclusion Wild-type HBx and the high proliferation-inducing mutant HBx can influence the miRNA expression profile of L02 cells.HBx down-regulates miR-338-3p and miR-55 1b in L02 cells,and the high proliferation-inducing mutant has a more robust effect.The mechanism of miR-338-3p- or miR-55 1b-mediated cell growth inhibition appears to be related to the direct modulation ofcyclinD1,cyclinG1,and E2F1.
