CAJ | 학술논문

目的研究survivin基因在肝癌细胞中的表达水平及survivin小干扰RNA对高侵袭潜能人肝癌细胞HCCLM6恶性表型的影响.方法收集4种人肝癌细胞株,以RT-PCR和Western blot检测survivin蛋白质表达,进而筛选出高表达survivin基因的肝癌细胞.构建针对survivin mRNA的干扰质粒pshRNA-survivin及阴性对照质粒pGPU6/GFP/Neo-NC,并将其转染HCCLM6细胞.实时荧光定量PCR检测survivin mRNA表达量的变化情况;肿瘤细胞黏附实验检测细胞黏附能力的改变及Matrigel侵袭实验检测细胞侵袭力的改变. 结果 4种肝癌细胞随侵袭能力升高survivin mRNA表达水平依次升高(P＜0.05),HCCLM6细胞survivin表达水平最高;Western blot结果与RT-PCR结果一致.HCCLM6细胞转染pshRNA-survivin后,survivin mRNA表达量明显下降,抑制率达93.500%±3.117%,与转染阴性对照质粒组(8.215%±0.797%)相比,差异有统计学意义(t=70.852,P＜0.01).细胞黏附实验结果显示,survivin重组质粒转染组黏附率为11.403%±1.256%,阴性对照组为32.545%±1.367%(t=20.732,P＜0.01).Matrigel侵袭实验结果显示,与阴性对照组[(32.6±1.4)]个相比,转染pshRNA-survivin后,细胞侵袭力明显下降[(13.5±0.9)个,t=14.5,P＜0.01].结论 Survivin与肝癌侵袭转移等恶性生物学行为有关,并随侵袭转移潜能升高表达水平升高.pshRNA-survivin可以特异性抑制高侵袭肝癌细胞HCCLM6中survivin表达,并显著降低其侵袭、黏附能力,小干扰RNA技术有望成为抑制肝癌侵袭转移的新途径.
목적 연구survivin기인재간암세포중적표체수평급survivin소간우RNA대고침습잠능인간암세포HCCLM6악성표형적영향.방법 수집4충인간암세포주,이RT-PCR화Western blot검측survivin단백질표체,진이사선출고표체survivin기인적간암세포.구건침대survivin mRNA적간우질립pshRNA-survivin급음성대조질립pGPU6/GFP/Neo-NC,병장기전염HCCLM6세포.실시형광정량PCR검측survivin mRNA표체량적변화정황;종류세포점부실험검측세포점부능력적개변급Matrigel침습실험검측세포침습력적개변. 결과 4충간암세포수침습능력승고survivin mRNA표체수평의차승고(P＜0.05),HCCLM6세포survivin표체수평최고;Western blot결과여RT-PCR결과일치.HCCLM6세포전염pshRNA-survivin후,survivin mRNA표체량명현하강,억제솔체93.500%±3.117%,여전염음성대조질립조(8.215%±0.797%)상비,차이유통계학의의(t=70.852,P＜0.01).세포점부실험결과현시,survivin중조질립전염조점부솔위11.403%±1.256%,음성대조조위32.545%±1.367%(t=20.732,P＜0.01).Matrigel침습실험결과현시,여음성대조조[(32.6±1.4)]개상비,전염pshRNA-survivin후,세포침습력명현하강[(13.5±0.9)개,t=14.5,P＜0.01].결론 Survivin여간암침습전이등악성생물학행위유관,병수침습전이잠능승고표체수평승고.pshRNA-survivin가이특이성억제고침습간암세포HCCLM6중survivin표체,병현저강저기침습、점부능력,소간우RNA기술유망성위억제간암침습전이적신도경.
Objectives To study survivin expression in human hepatoma cells and the effects of survivin siRNA on the malignant phenotypes of human hepatocellular cell line HCCLM6.Methods Four hepatocellular carcinoma(HCC)cell lines were used.Semi-quantitative RT-PCR and Westem blot were used to measure and compare their survivin expressions.The siRNA expression vector pshRNA-survivin targeting the mRNA of survivin and vector pGPU6/GFP/Neo-NC(as a control)were constructed.and then transfected into HCCLM6 cells.FQ-PCR was used to quantify the mRNA levels of survivin.The malignant phenotypes of transfected HCCLM6 cells,including invasive activities and adhesive capabilities,were analyzed.Results Survivin expression gradually increased with the increase of the invasion and metastasis behaviors of the four HCC cell lines (P＜0.05).The expression of survivin was highest in cell line HCCLM6.Survivin mRNA level was decreased bv 93.500%±3.117%after the pshRNA-survivin transfection.The cell adhesion rates significantly decreased in the cells transfected with pshRNA-survivin(cell adhesion rates were 11.403%±1.256%vs 32.545%±1.367%,t=20.732,P＜0.01).Themigrating numberofHCCLM6 cells(13.5±0.9)transfected with pshRNA-survivin was also significantly decreased(t=14.5,P＜0.01)as compared with the control group(32.6±1.4).Conclusion The expression of survivin in HCC might have a close relationship to their invasion and metastasis properties.Sequence-specific shRNA can significantly reduce the survivin expression in the HCCLM6 cell line.Suppression of survivin expression in HCCLM6 cells transfected with pshRNA-survivin can reduce their invasive and adhesive capabilities.