下调miR―21通过PDCD4抑制人增生性瘢痕成纤维细胞增殖并抑制PI3K-Akt信号通路.docVIP

下调miR―21通过PDCD4抑制人增生性瘢痕成纤维细胞增殖并抑制PI3K/Akt信号通路 　　[摘要]目的：探讨microRNA-21（miR-21）对人增生性瘢痕成纤维细胞中程序性细胞死亡因子（PDCD4）表达和细胞增殖的影响及机制。方法：利用荧光定量PCR检测正常皮肤成纤维细胞GM0639和人增生性瘢痕成纤维细胞（HSF）中miR-21的表达。构建含有PDCD4-pGC-Fu-GFP慢病毒和空载体对照慢病毒pGC-Fu-GFP感染HSF细胞。将HSF细胞按照不同处理方法分为：空白对照组、转染非特异miRNA的阴性对照组、转染miRNA-21抑制物的mir21 Inhibitor组、转染miR-21质粒的miR-21 mimic组、PDCD4-pGC-Fu-GFP组、pGC-Fu-GFP组和Ly294002组。转染后48h收集细胞，MTT检测细胞增殖情况。采用免疫荧光技术、荧光定量PCR和Western blot检测空白对照组、阴性对照组、miR-21 mimic组和miR-21 Inhibitor组细胞中PDCD4的表达以及基因和蛋白水平。采用荧光定量PCR和Western blot检测PDCD4-pGC-Fu-GFP组、pGC-Fu-GFP组和Ly294002组细胞周期相关蛋白C-MYC，CCND1以及通路相关蛋白p-Akt、JNK1的蛋白表达。结果：HSF 细胞中miR-21呈现高表达。与空白对照组相比，抑制miR-21表达可以抑制HSF细胞增殖，上调PDCD4表达。过表达PDCD4可以抑制细胞增殖，下调细胞周期相关蛋白C-MYC， CCND1的表达，同时抑制通路蛋白p-Akt和JNK1的表达。结论：下调miR-21的表达能够促进PDCD4表达，抑制HSF细胞增殖以及PI3K/AKt信号通路。 中国论文网 /6/view-7192982.htm 　　[关键词]microRNA-21；PDCD4；人增生性瘢痕成纤维细胞；细胞增殖；抑制；PI3K/Akt 　　[中图分类号]R619+.6 [文献标志码]A [文章编号]1008-6455（2015）23-0039-05 　　Abstract： Objective To study the effect and mechanism of microRNA-21 on the expression of PDCD4 and the proliferation of HSF cells. Methods The level of miR-21 in GM0639 and HSF cells was detected with qRT-PCR.HSF cells were divided into seven groups：control group，negative control group（transfected with no-related siRNA），miR21 Inhibitor group（transfected with miRNA-21 inhibitors），miR-21 mimic group（transfected with miR-21 mimic），PDCD4-pGC-Fu-GFP group，pGC-Fu-GFP group and Ly294002 group.The cells proliferation was detected by MTT after the transfection with 48h.The expression of PDCD4 gene and protein in the cells were detected by immunofluorescence，Real-time PCR and Western-blot methods respectively.The expression of C-MYC and CCND1 as well as p-Akt and JNK1 in the group of control，pGC-Fu-GFP and PDCD4-pGC-Fu-GFP were detected by Real-time PCR and Western blot，respectively. Results miR-21 was overexpressed in HSF cells.Compared with the group of control，down-regulation miR-21 obviously inhibited the HSF cells proliferation and induced the expression of PDCD4.PDCD4 overexpres