CAJ | 학술논문

目的构建polo样激酶-1(PLK1)基因小干扰RNA(siRNA),观察其对肝癌细胞凋亡的影响.方法设计合成3个PLK1 siRNA(siRNA1、siRNA2、siRNA3)干扰序列,构建干扰载体,转染人肝癌细胞株SMMC-7721,观察48 h后转染效率,采用实时定量逆转录-聚合酶链反应(RT-PCR)筛选下调PLK1 mRNA效果最好的siRNA；然后选择以该siRNA转染48 h的SMMC-7721细胞,分别以实时定量RT-PCR和Western blot法检测空白组、对照组及转染组细胞中PLK1基因和蛋白表达；流式细胞仪检测细胞周期及细胞凋亡的改变.结果肝癌细胞株SMMC-7721中存在PLK1蛋白的过表达.应用靶向PLK1的siRNA转染SMMC-7721细胞48 h后,PLK1 mRNA相对水平siRNA2组分别较无关对照组和空白对照组下降了74％和78％,而PLK1蛋白的表达siRNA2组分别较无关对照组和空白对照组下降了83％和88％,抑制率达88％；siRNA2组中出现大量凋亡细胞,与对照组比较差异有统计学意义(P＜ 0.05).结论 PLK1基因在肝癌细胞增殖中具有重要的调控作用.PLK1-siRNA转染可明显抑制癌细胞增殖,诱导凋亡是其重要机制之一.
목적 구건polo양격매-1(PLK1)기인소간우RNA(siRNA),관찰기대간암세포조망적영향.방법 설계합성3개PLK1 siRNA(siRNA1、siRNA2、siRNA3)간우서렬,구건간우재체,전염인간암세포주SMMC-7721,관찰48 h후전염효솔,채용실시정량역전록-취합매련반응(RT-PCR)사선하조PLK1 mRNA효과최호적siRNA；연후선택이해siRNA전염48 h적SMMC-7721세포,분별이실시정량RT-PCR화Western blot법검측공백조、대조조급전염조세포중PLK1기인화단백표체；류식세포의검측세포주기급세포조망적개변.결과 간암세포주SMMC-7721중존재PLK1단백적과표체.응용파향PLK1적siRNA전염SMMC-7721세포48 h후,PLK1 mRNA상대수평siRNA2조분별교무관대조조화공백대조조하강료74％화78％,이PLK1단백적표체siRNA2조분별교무관대조조화공백대조조하강료83％화88％,억제솔체88％；siRNA2조중출현대량조망세포,여대조조비교차이유통계학의의(P＜ 0.05).결론 PLK1기인재간암세포증식중구유중요적조공작용.PLK1-siRNA전염가명현억제암세포증식,유도조망시기중요궤제지일.
Objective To study effects of polo-like kinase-1 (PLK1) small interfering RNA (siRNA) on proliferation of human hepatocellular carcinoma (HCC) cells.Methods Three PLK1 siRNAs (siRNA1,siRNA2,siRNA3) were constructed and use to transfect SMMC-7721 cells.Transfection efficiency at 48 h was observed.Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to find the most effective siRNA which was then used to transfect SMMC-7721 cells.RT-PCR and Western blotting were used to detect PLK1 expression in SMMC-7721 cells which were divided into different groups.Flow cytometry (FCM) was used to detect apoptosis rate and phase distribution of cell cycles.Results PLK1 protein was overexpressed in SMMC-7721 cells.SMMC-7721 cells transfected with low doses of siRNAs targeted against PLK1 had greatly decreased levels of PLK1 mRNA and protein.Compared to blank control and negative control 48 h after transfection,siRNA2 reduced PLK1 mRNA by 74％ and 78％,and PLK1 protein by 83％ and 88％,respectively.Apoptosis rate was increased remarkably and the phenotypes of apoptosis could be seen in transfected cells 48 h after transfection.Conclusion PLK1 may play an important role in proliferation of HCC cells.PLK1 siRNA transfection can inhibit proliferation of HCC cells through apoptosis induction.