摘要
目的:观察微小RNA(miR)-144对前列腺癌细胞增殖侵袭的影响及其对转化生长因子-β(TGF-β)/Smad信号通路的影响。方法:采用实时荧光定量聚合酶链反应(qRT-PCR)检测LNCaP、PC-3、SW1990人前列腺癌细胞和RWPE-1人正常前列腺上皮细胞中miR-144的表达水平;以miR-144表达量最低的PC-3细胞和表达量最高的LNCaP细胞作为研究对象,转染miR-144 mimics-NC序列为NC组,转染miR-144 mimics序列为miR-144过表达(mimics)组,荧光显微镜观察转染情况;qRTPCR检测转染后miR-144的表达水平,细胞活力检测试剂盒(CCK-8)检测细胞增殖,Transwell实验检测细胞侵袭能力,生物信息学预测及荧光素酶报告基因检验miR-144的潜在靶基因,蛋白免疫吸附法(Western Blot)检测TGF-β/Smad通路蛋白表达水平。结果:与正常前列腺上皮细胞RWPE-1比较,LNCaP、PC-3、SW1990前列腺癌细胞中miR-144的表达水平均显著降低(P<0.05),miR-144 mimics转染后细胞转染效率均达到90%以上。与NC组比较,mimics组细胞中miR-144的相对表达水平显著升高,细胞的增殖活性和侵袭能力显著降低(P<0.05或P<0.01)。与TGF-β-Wt+miR-144 mimics-NC共转染组比较,TGF-β-Wt+miR-144 mimics转染组荧光素酶活性显著降低(P<0.05)。TGF-β转录本3’UTR区域具有miR-144的潜在靶点,miR-144潜在靶基因为TGF-β,在24~120 h,与miR-144 mimics-NC组相比,miR-144 mimics、miR-144 mimics+TGF-β组细胞活性显著降低(P<0.05);与miR-144 mimics组相比,miR-144 mimics+TGF-β组细胞活性显著升高(P<0.05)。与NC组比较,mimics组细胞中p-Smad2/Smad2、p-Smad3/Smad3水平及TGF-β、TGF-βR蛋白表达水平显著降低(P<0.01)。结论:miR-144可能通过TGF-β/Smad信号通路抑制前列腺癌细胞的增殖及侵袭。
Objective: To observe the effects of micro(miR)-144 on the proliferation and invasion of prostate cancer cells through transforming growth factor-β(TGF-β)/Smad signaling pathway. Methods: The expression levels of miR-144 in LNCaP,PC-3,SW1990 human prostate cancer cells and RWPE-1 human normal prostate epithelial cells were detected by qRT-PCR. The PC-3 cells with the lowest expression of miR-144 and the LNCaP cells with the highest expression were the research objects,those transfected with miR-144 mimics-NC sequence were used as NC group,and those transfected with miR-144 mimics sequence were used as mimimics group. The cell transfection was observed by fluorescence microscopy,the expression of miR-144 after transfection was detected by qRT-PCR,the proliferation of PC-3 cells was detected by CCK-8 assay,and transwell assay was used to detect cell invasion,bioinformatics prediction and luciferase report gene were used to report potential target genes of miR-144,and Western blot was used to detect the expression of TGF-β/Smad pathway protein. Results: Compared with those in the normal prostate epithelial cells RWPE-1,the expression levels of miR-144 in LNCaP,PC-3 and SW1990 prostate cancer cells were significantly reduced(P<0.05),and the transfection efficiency of miR-144 mimics after transfection reached more than 90%. Compared with that in NC group,the relative expression level of miR-144 in mimics group was significantly increased,and the proliferation activity and invasion ability of cells were significantly reduced(P<0.05 or P<0.01). Compared with TGF-β-wt+mir-144 mimics NC co-transfection group,the luciferase activity of TGF-β-wt+mir-144 mimics transfection group was significantly lower(P<0.05).TGF-β transcript 3’UTR region had the potential target of miR-144,the potential target gene of miR-144 was TGF-β,and during 24 h and 120 h,compared with that in miR-144 mimics-NC group,the cell activity in miR-144 mimics group and miR-144 mimics+TGF-β group decreased(P<0.05). Compared with that in miR-144 mimics group,the cell activity in miR-144 mimics+TGF-β group increased(P<0.05). Compared with those in NC group,the expression levels of p-Smad2/Smad2 and p-Smad3/Smad3,the protein expression levels of TGF-β and TGF-β R in the cells of miR-144 overexpression group were significantly reduced(P<0.01). Conclusion: MiR-144 may inhibit the proliferation and invasion of prostate cancer cells through TGF-β/Smad signaling pathway.
作者
王辉
冯建明
高卫军
李占琦
Wang Hui;Feng Jianmin;Gao Weijun;Li Zhanqi(Department of Urology,No.215 Hospital of Shaanxi Nuclear Industry,Shaanxi Xianyang 712000,China)
出处
《中国药师》
CAS
2021年第2期282-287,共6页
China Pharmacist
