摘要
目的探讨转录因子叉头框蛋白O3a(FOXO3a)对前列腺癌细胞上皮间质转化(EMT)的影响及其作用机制。方法采用逆转录聚合酶链式反应(RT-PCR)检测正常前列腺上皮细胞RWPE-1和前列腺癌细胞LNCaP、DUl45、PC-3中FOXO3a表达水平,选择FOXO3a表达最低的前列腺癌细胞系转染pcDNA-FOXO3a、pcDNA-NC构建FOXO3a过表达细胞系(pc-FOXO3a)和阴性对照(NC),采用MTT法、流式细胞法检测细胞活性和凋亡情况,Transwell试验、划痕试验检测细胞侵袭和迁移能力,蛋白质印迹法(Western blotting)检测钙黏附蛋白E(E-cadherin)、波形蛋白(Vimentin)、神经型钙黏附蛋白(N-cadherin)、Snail、c-Myc、细胞周期蛋白D1(cyclin D1)、剪切型胱天蛋白酶-3(Cleaved-Cas-3)、Bcl-2相关X(Bax)、B细胞淋巴瘤-2(Bcl-2)、β-连环蛋白(β-catenin)、Wnt1水平。运用氯化锂(Wnt/β-catenin信号通路激活剂)作用于pcDNA-FOXO3a前列腺细胞,分析细胞活性、凋亡和上皮间质转换(EMT)情况。结果LNCaP、DUl45、PC-3细胞中FOXO3a表达水平均显著低于RWPE-1细胞[(0.23±0.04)、(0.53±0.05)、(0.42±0.06)比(1.02±0.08)](P<0.05),其中LNCaP细胞中FOXO3a表达水平最低。pc-FOXO3a LNCaP细胞活性、单个视野细胞侵袭数目、划痕迁移率显著低于NC LNCaP细胞(P<0.05),细胞凋亡率显著高于NC LNCaP细胞(P<0.05);pc-FOXO3a LNCaP细胞N-cadherin、Vimentin、Snail、β-catenin、c-Myc、cyclin D1、Bcl-2、Wnt1水平显著低于NC LNCaP细胞(P<0.05),而E-cadherin、Cleaved-Cas-3、Bax水平显著高于NC LNCaP细胞(P<0.05)。LiCl-pc-FOXO3a LNCaP细胞活性、单个视野细胞侵袭数目、划痕迁移率、N-cadherin、Vimentin、Snail水平显著高于LiCl-pc-FOXO3a-Ctrl LNCaP细胞(P<0.05),细胞凋亡率、E-cadherin水平显著低于LiCl-pc-FOXO3a-Ctrl LNCaP细胞(P<0.05)。结论FOXO3a表达升高可降低前列腺癌细胞活性、促进细胞凋亡,抑制细胞EMT,可能与抑制Wnt/β-catenin信号通路活化有关。
Objective To explore the effects and action mechanism of forkhead box protein O3a(FOXO3a)on epithelial-mesenchymal transition(EMT)of prostate cancer cells.Methods The expression levels of FOXO3a in normal prostate epithelial cells(RWPE-1)and prostate cancer cells(LNCaP,DU145,PC-3)were detected by RT-PCR.The prostate cancer cell line with the lowest FOXO3a expression was selected and was transfected with pcDNA-FOXO3a and pcDNA-NC to construct FOXO3a overexpression cell line(pc-FOXO3a)and negative control(NC).The cell viability and apoptosis were detected by MTT and flow cytometry.The invasion and migration of cells were detected by Transwell assay and scratch assay.The levels of E-cadherin,Vimentin,N-cadherin,Snail,c-Myc,cyclin D1,Cleaved-Cas-3,Bax,Bcl-2,β-catenin and Wnt1 were detected by Western blot.The pcDNA-FOXO3a prostate cells were treated with lithium chloride(Wnt/β-catenin signaling pathway activator)to analyze cell viability,apoptosis and EMT.Results The expression level of FOXO3a in LNCaP,DU145 and PC-3 cells was significantly lower than that in RWPE-1 cells[(0.23±0.04),(0.53±0.05),(0.42±0.06)vs.(1.02±0.08)](P<0.05),and the expression level of FOXO3a in LNCaP cells was the lowest.The cells activity,number of invasion cells in per field and migration rate of scratches in pc-FOXO3a LNCaP cells were significantly lower than those in NC LNCaP cells(P<0.05),while apoptosis rate was significantly higher than that in NC LNCaP cells(P<0.05).The levels of N-cadherin,Vimentin,Snail,β-catenin,c-Myc,cyclin D1,Bcl-2 and Wnt1 in pc-FOXO3a LNCaP cells were significantly lower than those in NC LNCaP cells(P<0.05),while the levels of E-cadherin,Cleaved-Cas-3 and Bax were significantly higher than those in NC LNCaP cells(P<0.05).The cells activity,number of invasion cells in per field,migration rate of scratches,and levels of N-cadherin,Vimentin and Snail in LiCl-pcFOXO3a LNCaP cells were significantly higher than those in LiCl-pc-FOXO3a-Ctrl LNCaP cells(P<0.05),while apoptosis rate and Ecadherin level were significantly lower than those in LiCl-pc-FOXO3a-Ctrl LNCaP cells(P<0.05).Conclusion Increased expression of FOXO3a can reduce the viability of prostate cancer cells,promote apoptosis,and inhibit EMT,which may be related to inhibiting the activation of Wnt/β-catenin signaling pathway.
作者
夏明亮
任川川
李云龙
朱海松
张超
李军
XIA Mingliang;REN Chuanchuan;LI Yunlong;ZHU Haisong;ZHANG Chao;LI Jun(Department of Urinary Surgery,Zhumadian Central Hospital,Zhumadian,Henan 463000,China;Department of Urinary Surgery,The First Affiliated Hospital of Zhengzhou University,Zhengzhou,Henan 450000,China)
出处
《安徽医药》
CAS
2022年第2期297-303,共7页
Anhui Medical and Pharmaceutical Journal