中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2014年
6期
1225-1227
,共3页
顾鹏%周毅彬%阳东荣%单玉喜%薛波新
顧鵬%週毅彬%暘東榮%單玉喜%薛波新
고붕%주의빈%양동영%단옥희%설파신
前列腺肿瘤%基质交联分子1%肿瘤转移%上皮-间充质转化
前列腺腫瘤%基質交聯分子1%腫瘤轉移%上皮-間充質轉化
전렬선종류%기질교련분자1%종류전이%상피-간충질전화
Prostate cancer%Stormal interaction molecule 1%Tumor metastasis%Epithelial-mesenchymal transition
目的 观察基质交联分子1(STIM1)对前列腺癌DU145细胞迁移、侵袭的影响并探讨其在上皮-间充质转化(EMT)过程中的作用.方法 将携带STIM1基因的小干扰RNA(siRNA)慢病毒载体STIM1-pGCSIL-GFP转染人激素非依赖性前列腺癌DU145细胞(KD组),同时设阴性对照组(NC组)及空白对照组(CON组).逆转录-聚合酶链反应(RT-PCR)及Western blot验证抑制STIM1表达的有效性.采用划痕实验及Transwell迁移、侵袭实验检测抑制STIM1对肿瘤细胞运动及迁移、侵袭能力的影响.采用半定量RT-PCR及Western blot分别在mRNA及蛋白水平检测EMT标志物E-钙黏蛋白(E-cadherin)、波动蛋白(Vimentin)、α-平滑肌肌动蛋白(α-SMA)在各组细胞中的差异.结果 与对照组比较,转染STIM1-pGCSIL-GFP后,DU145细胞内STIM1的mRNA及蛋白水平均明显降低.划痕实验显示KD组DU145细胞的运动能力明显降低.细胞迁移结果显示,CON、NC、KD组每个视野细胞数分别为(92.0±14.2)、(87.3±2.3)、(60.8±10.3)个,KD组数目明显减少(P＜0.05);细胞侵袭实验提示,CON、NC、KD组每视野细胞数分别为(79.6±3.7)、(80.0±3.5)、(45.8±5.8)个,KD组侵袭数目明显减少(P＜0.05).RT-PCR和Western blot结果显示,KD组上皮标志物E-cadherin mRNA及蛋白表达水平增高(分别为NC组的1.38、1.72倍);间质标志物Vimentin的mRNA及蛋白表达水平降低(分别为NC组的0.67、0.74倍),α-SMA mRNA及蛋白表达水平降低(分别为NC组的0.23、0.54倍,P＜0.05).结论 抑制前列腺癌DU145细胞中STIM1表达后,细胞运动及迁移、侵袭能力均明显降低,这可能与逆转EMT相关.
目的 觀察基質交聯分子1(STIM1)對前列腺癌DU145細胞遷移、侵襲的影響併探討其在上皮-間充質轉化(EMT)過程中的作用.方法 將攜帶STIM1基因的小榦擾RNA(siRNA)慢病毒載體STIM1-pGCSIL-GFP轉染人激素非依賴性前列腺癌DU145細胞(KD組),同時設陰性對照組(NC組)及空白對照組(CON組).逆轉錄-聚閤酶鏈反應(RT-PCR)及Western blot驗證抑製STIM1錶達的有效性.採用劃痕實驗及Transwell遷移、侵襲實驗檢測抑製STIM1對腫瘤細胞運動及遷移、侵襲能力的影響.採用半定量RT-PCR及Western blot分彆在mRNA及蛋白水平檢測EMT標誌物E-鈣黏蛋白(E-cadherin)、波動蛋白(Vimentin)、α-平滑肌肌動蛋白(α-SMA)在各組細胞中的差異.結果 與對照組比較,轉染STIM1-pGCSIL-GFP後,DU145細胞內STIM1的mRNA及蛋白水平均明顯降低.劃痕實驗顯示KD組DU145細胞的運動能力明顯降低.細胞遷移結果顯示,CON、NC、KD組每箇視野細胞數分彆為(92.0±14.2)、(87.3±2.3)、(60.8±10.3)箇,KD組數目明顯減少(P＜0.05);細胞侵襲實驗提示,CON、NC、KD組每視野細胞數分彆為(79.6±3.7)、(80.0±3.5)、(45.8±5.8)箇,KD組侵襲數目明顯減少(P＜0.05).RT-PCR和Western blot結果顯示,KD組上皮標誌物E-cadherin mRNA及蛋白錶達水平增高(分彆為NC組的1.38、1.72倍);間質標誌物Vimentin的mRNA及蛋白錶達水平降低(分彆為NC組的0.67、0.74倍),α-SMA mRNA及蛋白錶達水平降低(分彆為NC組的0.23、0.54倍,P＜0.05).結論 抑製前列腺癌DU145細胞中STIM1錶達後,細胞運動及遷移、侵襲能力均明顯降低,這可能與逆轉EMT相關.
목적 관찰기질교련분자1(STIM1)대전렬선암DU145세포천이、침습적영향병탐토기재상피-간충질전화(EMT)과정중적작용.방법 장휴대STIM1기인적소간우RNA(siRNA)만병독재체STIM1-pGCSIL-GFP전염인격소비의뢰성전렬선암DU145세포(KD조),동시설음성대조조(NC조)급공백대조조(CON조).역전록-취합매련반응(RT-PCR)급Western blot험증억제STIM1표체적유효성.채용화흔실험급Transwell천이、침습실험검측억제STIM1대종류세포운동급천이、침습능력적영향.채용반정량RT-PCR급Western blot분별재mRNA급단백수평검측EMT표지물E-개점단백(E-cadherin)、파동단백(Vimentin)、α-평활기기동단백(α-SMA)재각조세포중적차이.결과 여대조조비교,전염STIM1-pGCSIL-GFP후,DU145세포내STIM1적mRNA급단백수평균명현강저.화흔실험현시KD조DU145세포적운동능력명현강저.세포천이결과현시,CON、NC、KD조매개시야세포수분별위(92.0±14.2)、(87.3±2.3)、(60.8±10.3)개,KD조수목명현감소(P＜0.05);세포침습실험제시,CON、NC、KD조매시야세포수분별위(79.6±3.7)、(80.0±3.5)、(45.8±5.8)개,KD조침습수목명현감소(P＜0.05).RT-PCR화Western blot결과현시,KD조상피표지물E-cadherin mRNA급단백표체수평증고(분별위NC조적1.38、1.72배);간질표지물Vimentin적mRNA급단백표체수평강저(분별위NC조적0.67、0.74배),α-SMA mRNA급단백표체수평강저(분별위NC조적0.23、0.54배,P＜0.05).결론 억제전렬선암DU145세포중STIM1표체후,세포운동급천이、침습능력균명현강저,저가능여역전EMT상관.
Objective To observe the effect of stormal interaction molecule 1 (STIM1) on cell migration and invasion in prostate cancer DU145 cells,and explore whether STIM1 contribute to reverse the epithelial-mesenchymal transition (EMT) transformation.Methods DU145 cells were transfected with lentiviral vector STIM1-pGCSIL-GFP (KD group) and related negative vector (NC group),normal DU145 cells without any interference were used as control (CON group).After transfection,the expression of STIM1 in DU145 cells was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting.The cell mobility,migration and invasion ability of DU145 cells were respectively measured by wound-healing assay and transwell migration and invasion assays.The expression of EMT markers [E-cadherin,Vimentin,α-smooth muscle actin (ct-SMA)] were detected by semi-quantitative RT-PCR and Western blotting analysis.Results After transfected with STIM1-pGCSIL-GFP,STIM1 expression in DU145 cells was decreased at both mRNA and protein levels (P ＜ 0.05),and the cell mobility was decreased.The number of migrated cells in group CON,NC and KD were 92.0 ± 14.2,87.3 ± 2.3,60.8 ± 10.3,respectively.The number of invaded cells in group CON,NC and KD were 79.6 ± 3.7,80.0 ± 3.5,45.8 ± 5.8,respectively.These indicated that cell migration and invasion ability of transfected cells were decreased (P ＜ 0.05).Epithelial marker E-cadherin increased and mesenchymal markers Vimentin,α-SMA decreased in transfected cells that detected by both RT-PCR and Western blotting analysis (P ＜ 0.05).Conclusion STIM1 inhibition could decrease the cell mobility,cell migration and invasion.This phenomenon may due to STIM1 could reverse the EMT transformation in DU145 cells.
