CAJ | 학술논문

目的观察生长抑素受体-2(SSTR2)基因对肝癌细胞株MHCC-97H侵袭转移及上皮-间充质转化(EMT)特性的影响.方法构建慢病毒3FLAG-puromycin-LV及SSTR2-LV,转染肝癌细胞株MHCC-97H,噻唑蓝(MTT)、免疫荧光检测转染效率.采用Transwell侵袭实验和迁移实验分别检测未转染细胞(空白对照组)、转染3FLAG-puromycin-LV细胞(阴性对照组)和转染SSTR2-LV细胞(实验组)侵袭转移能力的强弱.镜下观察转染前后细胞形态的变化,免疫荧光检测转染前后细胞上皮表型蛋白和间质表型蛋白的定位和表达,实时定量聚合酶链反应(Real-time PCR)检测转染后细胞SSTR2的表达,Westem blot检测E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)蛋白的表达.结果 SSTR2-LV可以有效转染肝癌细胞株MHCC-97H,稳定表达SSTtR2的mRNA和蛋白.转染SSTR2-LV的肝癌细胞株MHCC-97H较空白对照组和阴性对照组侵袭迁移能力明显受到抑制(P＜0.05).镜下观察肝癌细胞株MHCC-97H转染SSTR2-LV后,由成纤维细胞样、排列分散,转变为细胞排列紧密如铺路石.免疫荧光染色结果可见肝癌细胞株MHCC-97H转染后细胞膜表达的E-cadherin荧光由胞质转移至细胞周边浓聚,而Vimentin的荧光强度较前下降.采用Western blot进行蛋白定量分析,转染后MHCC-97H细胞上皮表型标志性蛋白E-cadherin、β-连环蛋白(β-catenin)表达量增高,间质表型标志性蛋白N-cadherin、Vimentin表达量减少,差异具有统计学意义(P＜0.05).结论转染再表达SSTR2MHCC-97H细胞可以增加上皮表型蛋白表达,减少间质表型蛋白,从而逆转肝癌细胞EMT,导致肿瘤细胞侵袭能力减弱.
목적 관찰생장억소수체-2(SSTR2)기인대간암세포주MHCC-97H침습전이급상피-간충질전화(EMT)특성적영향.방법 구건만병독3FLAG-puromycin-LV급SSTR2-LV,전염간암세포주MHCC-97H,새서람(MTT)、면역형광검측전염효솔.채용Transwell침습실험화천이실험분별검측미전염세포(공백대조조)、전염3FLAG-puromycin-LV세포(음성대조조)화전염SSTR2-LV세포(실험조)침습전이능력적강약.경하관찰전염전후세포형태적변화,면역형광검측전염전후세포상피표형단백화간질표형단백적정위화표체,실시정량취합매련반응(Real-time PCR)검측전염후세포SSTR2적표체,Westem blot검측E-개점단백(E-cadherin)、N-개점단백(N-cadherin)、파형단백(Vimentin)단백적표체.결과 SSTR2-LV가이유효전염간암세포주MHCC-97H,은정표체SSTtR2적mRNA화단백.전염SSTR2-LV적간암세포주MHCC-97H교공백대조조화음성대조조침습천이능력명현수도억제(P＜0.05).경하관찰간암세포주MHCC-97H전염SSTR2-LV후,유성섬유세포양、배렬분산,전변위세포배렬긴밀여포로석.면역형광염색결과가견간암세포주MHCC-97H전염후세포막표체적E-cadherin형광유포질전이지세포주변농취,이Vimentin적형광강도교전하강.채용Western blot진행단백정량분석,전염후MHCC-97H세포상피표형표지성단백E-cadherin、β-련배단백(β-catenin)표체량증고,간질표형표지성단백N-cadherin、Vimentin표체량감소,차이구유통계학의의(P＜0.05).결론 전염재표체SSTR2MHCC-97H세포가이증가상피표형단백표체,감소간질표형단백,종이역전간암세포EMT,도치종류세포침습능력감약.
Objective To investigate the effect of the somatostatin receptor subtype 2 (SSTR2) on the migration and invasion of human Hepatocellular carcinoma cells MHCC-97H and the change of epithelial-mesenchymal transition (EMT).Methods Constructed 3FLAG-puromycin-LV and SSTR2-LV and transfected hunman Hepatocellular carcinoma cells (MHCC-97H) respectively.The expression of SSTR2 was detected by real-time quantitative polymerase chain reaction (Real-time PCR) and Western blot.The migration and invasion of Non-transfected (blank control group),3FLAG-puromycin-LV transfected (negative control group) and SSTR2-LV (experimental group) transfected MHCC-97H cells was detected by Transwell invasion assay and Transwell migration assay.The changes in morphology of cells were observed by optical microscopy.The localization and expression of the epithelial markers and the mesenchymal markers were assayed by immunofluorescence and Western blotting.Results The stable mRNA and protein expression of SSTR2 was detected in the cells transfected with SSTR2-LV.The migration and invasion was restrained in the experimental group compared with control groups (P ＜ 0.05).Transfection of SSTR2-LV induced a morphologic change of MHCC-97H cells from fibroblastic shape with cell scattering to a paving stone structure with cell-cell adhesion.In SSTR2-LV-transfected cells,the localization of E-cadherin was altered from the cytoplasm to cell-cell contacts and the level of Vimentin decreased.Moreover,the expression of the epithelial marker,E-cadherin and β-catenin,were increased; the expression of the mesenchymal marker,N-cadherin and Vimentin,were reduced.Conclusion MHCC-97H re-expression of SSTR2 induced the increasing expression of epithelial marker and the decrease expression of mesenchymal marker reversed HCC EMT then suppressed the HCC cells migration and invasion.