中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2010年
16期
1131-1136
,共6页
罗勇%贺大林%姜永光%宁亮%申树林%赵佳晖%崔新浩
囉勇%賀大林%薑永光%寧亮%申樹林%趙佳暉%崔新浩
라용%하대림%강영광%저량%신수림%조가휘%최신호
前列腺肿瘤%上皮细胞间质转化态%β-连环素%缺氧诱导因子-1α
前列腺腫瘤%上皮細胞間質轉化態%β-連環素%缺氧誘導因子-1α
전렬선종류%상피세포간질전화태%β-련배소%결양유도인자-1α
Prostate neoplasms%Epithelial-mesenchynml transition%beta catenin%HIF-1α
目的 明确β-连环蛋白(catenin)信号通路是否参与缺氧诱导因子(HIF)-1α诱导人前列腺癌细胞发生上皮细胞间质转化态(EMT)转化过程.方法 应用Western印迹法检测HIF-1α、Glut-1和VEGF蛋白表达,确认2种新构建前列腺癌细胞株(LNCaP/HIF1α和PC-3/HIF1α)的稳定性;然后应用Western印迹法检测EMT指标蛋白(E-cadherin、CK18、Vimentin、N-cadherin及Fibronetin)的表达,对5种人前列腺癌细胞株(LNCaP、LNCaP/HIF1α、PC-3、PC-3/HIF1α和IA8)的EMT特性进行鉴定;进一步应用Transwell和MTT技术检测5种细胞株的体外侵袭和增殖潜能;最后,用RT-PCR和Western印迹法检测5种EMT特性不同的细胞株中β-catenin、tGSK-3β和pGSK-3β的表达,总结分析该信号通路活性与细胞EMT特性的关联.结果 (1)LNCaP/HIF1α和PC-3/HIF1α中出现明显的HIF-1α蛋白条带,同时Glut-1和VIEGF表达呈强阳性;(2)Pc-3、LNCaP和Pc-3/HIF1α是EMT阴性细胞,而LNCaP/HIF1α和IA8是EMT阳性细胞;(3)PC-3/HIF1α和LNCaP/HIF1α、IA8体现出了较PC-3和LNCaP更为强大的体外侵袭和增殖潜能;(4)与LNCaP和PC-3相比,PC-3/HIF1α和LNCaP/HIF1α、IA8中tGSK-3β和pGSK-3β的蛋白表达相对减少,但p-GSK3β/t-GSK3β比值相应较高,β-catenin蛋白表达在LNCaP/HIF1α和IA8中相对较高,PC-3/HIF1α却表达较低;基因检测结果与前述蛋白表达规律基本一致,但是与β-catenin蛋自在PC-3/HIF1α中低表达不吻合的是,PC-3/HIF1α中β-catenin mBNA水平与其在LNCaP/HIF1α和IA8中一样呈现出强表达特点.结论 β-catenin信号通路的活性状态与细胞EMT特性及其体外侵袭和增殖潜能有密切关系,该信号通路可能是介导HIF-1α诱导人前列腺癌细胞EMT过程的重要"桥梁".
目的 明確β-連環蛋白(catenin)信號通路是否參與缺氧誘導因子(HIF)-1α誘導人前列腺癌細胞髮生上皮細胞間質轉化態(EMT)轉化過程.方法 應用Western印跡法檢測HIF-1α、Glut-1和VEGF蛋白錶達,確認2種新構建前列腺癌細胞株(LNCaP/HIF1α和PC-3/HIF1α)的穩定性;然後應用Western印跡法檢測EMT指標蛋白(E-cadherin、CK18、Vimentin、N-cadherin及Fibronetin)的錶達,對5種人前列腺癌細胞株(LNCaP、LNCaP/HIF1α、PC-3、PC-3/HIF1α和IA8)的EMT特性進行鑒定;進一步應用Transwell和MTT技術檢測5種細胞株的體外侵襲和增殖潛能;最後,用RT-PCR和Western印跡法檢測5種EMT特性不同的細胞株中β-catenin、tGSK-3β和pGSK-3β的錶達,總結分析該信號通路活性與細胞EMT特性的關聯.結果 (1)LNCaP/HIF1α和PC-3/HIF1α中齣現明顯的HIF-1α蛋白條帶,同時Glut-1和VIEGF錶達呈彊暘性;(2)Pc-3、LNCaP和Pc-3/HIF1α是EMT陰性細胞,而LNCaP/HIF1α和IA8是EMT暘性細胞;(3)PC-3/HIF1α和LNCaP/HIF1α、IA8體現齣瞭較PC-3和LNCaP更為彊大的體外侵襲和增殖潛能;(4)與LNCaP和PC-3相比,PC-3/HIF1α和LNCaP/HIF1α、IA8中tGSK-3β和pGSK-3β的蛋白錶達相對減少,但p-GSK3β/t-GSK3β比值相應較高,β-catenin蛋白錶達在LNCaP/HIF1α和IA8中相對較高,PC-3/HIF1α卻錶達較低;基因檢測結果與前述蛋白錶達規律基本一緻,但是與β-catenin蛋自在PC-3/HIF1α中低錶達不吻閤的是,PC-3/HIF1α中β-catenin mBNA水平與其在LNCaP/HIF1α和IA8中一樣呈現齣彊錶達特點.結論 β-catenin信號通路的活性狀態與細胞EMT特性及其體外侵襲和增殖潛能有密切關繫,該信號通路可能是介導HIF-1α誘導人前列腺癌細胞EMT過程的重要"橋樑".
목적 명학β-련배단백(catenin)신호통로시부삼여결양유도인자(HIF)-1α유도인전렬선암세포발생상피세포간질전화태(EMT)전화과정.방법 응용Western인적법검측HIF-1α、Glut-1화VEGF단백표체,학인2충신구건전렬선암세포주(LNCaP/HIF1α화PC-3/HIF1α)적은정성;연후응용Western인적법검측EMT지표단백(E-cadherin、CK18、Vimentin、N-cadherin급Fibronetin)적표체,대5충인전렬선암세포주(LNCaP、LNCaP/HIF1α、PC-3、PC-3/HIF1α화IA8)적EMT특성진행감정;진일보응용Transwell화MTT기술검측5충세포주적체외침습화증식잠능;최후,용RT-PCR화Western인적법검측5충EMT특성불동적세포주중β-catenin、tGSK-3β화pGSK-3β적표체,총결분석해신호통로활성여세포EMT특성적관련.결과 (1)LNCaP/HIF1α화PC-3/HIF1α중출현명현적HIF-1α단백조대,동시Glut-1화VIEGF표체정강양성;(2)Pc-3、LNCaP화Pc-3/HIF1α시EMT음성세포,이LNCaP/HIF1α화IA8시EMT양성세포;(3)PC-3/HIF1α화LNCaP/HIF1α、IA8체현출료교PC-3화LNCaP경위강대적체외침습화증식잠능;(4)여LNCaP화PC-3상비,PC-3/HIF1α화LNCaP/HIF1α、IA8중tGSK-3β화pGSK-3β적단백표체상대감소,단p-GSK3β/t-GSK3β비치상응교고,β-catenin단백표체재LNCaP/HIF1α화IA8중상대교고,PC-3/HIF1α각표체교저;기인검측결과여전술단백표체규률기본일치,단시여β-catenin단자재PC-3/HIF1α중저표체불문합적시,PC-3/HIF1α중β-catenin mBNA수평여기재LNCaP/HIF1α화IA8중일양정현출강표체특점.결론 β-catenin신호통로적활성상태여세포EMT특성급기체외침습화증식잠능유밀절관계,해신호통로가능시개도HIF-1α유도인전렬선암세포EMT과정적중요"교량".
Objective Epithelial-mesenchymal transition(EMT)is an important process in tumor development.Several studies suggest that the β-catenin signal pathway may play an important role in EMT.However,there is no direct evidence showing that this pathway actually determines the EMT induced by exogenous signal.Our previous study has successfully proved that over-expression of HIF-1α could induce EMT in LNCaP cells.but not in Pc-3.So the present study was intended to indicate that the signal of HIF1αfor inducing prostate cancer cell to undergo EMT mish pass through the β-catenin pathway.Methods Firstly,we analyzed the expression of HIF-1αand its target proteins in LNCaP/HIF1α and PC-3/HIF1α by Western blot.And then EMT-associated proteins were detected by Western blot. Furthermore the potency of invasiveness and proliferation of several cell lines were evaluated by transwell and MTT assay.Lastly the expressions of β-catenin and GSK-3βin these cells were analyzed by Western blot and RT-PCR. Results HIF-1α,Glut-1 and、VEGF were highly expressed in LNCaP/HIF1α and PC-3/HIF1α And PC-3,LNCaP and PC-3/HIF1αwere EMT-negative cell lines whereas LNCaP/HIF1α and IA8 had undergone EMT