摘要
目的观察微小RNA（miRNA，miR）-188-5p在前列腺癌中的表达和对前列腺癌细胞增殖和转移的生物学影响。方法采用实时定量反转录聚合酶链反应（RT-qPCR）技术检测miR-188-5p在前列腺癌组织和前列腺癌细胞中的表达。将miR．188-5pmimic利用Lipfectamine2000转染至前列腺癌细胞，通过软琼脂细胞克隆形成实验、前列腺癌细胞侵袭和迁移实验研究前列腺癌细胞生物功能的改变。利用生物信息学软件miRanda预测miR-188-5p的靶基因。采用双荧光素酶报告实验验证miR-188-5p对靶基因的直接调控。结果miR-188-5pmRNA水平在前列腺癌组织中与前列腺癌旁组织和前列腺增生组织比较表达下调（0．996±0．024、0．990±0．094、0．385±0．031），差异有统计学意义（P〈0．05）。miR-188-5pmRNA水平在LNCaP、DU145和PC-3细胞中较RWPE-1明显下降（1．022±0．012、0．409±0．034、0．358±0．024、0．255±0．018），且差异有统计学意义（P〈0．01）。LNCaP和PC-3细胞中miR-188-5pmimic转染组细胞的体外克隆形成能力明显低于空白对照组（21±5、47±3；25±4、51±5），差异有统计学意义（P〈0．01）。在细胞侵袭和迁移实验中，miR-188-5pmimic转染组PC-3和LNCaP细胞到达对侧的迁移细胞数明显低于空白对照组，差异有统计学意义（P〈0．05）。利用生物信息学软件miranda预测溶酶体相关4次跨膜蛋白质β（LAPTM4B）基因是miR-188-5p可能作用的靶基因。双荧光素酶报告实验确定miR-188-5p与LAPTM4B之间的直接调控关系。结论miR-188-5p在前列腺癌组织和细胞中表达显著下调，过表达miR-188-5p可以抑制前列腺癌细胞的增殖和转移能力。miR-188-5p可能通过作用靶基因LAPTM4B参与调控前列腺癌的生长和转移。
Objective To measure the expression level of microRNA （miRNA, miR） -188 -5p in prostate carcinoma （PCa） and investigate its biological functions on prostate carcinoma cell lines. Methods In 30 PCa and 40 benign prostatic hyperplasia （BPH） samples, quantifieational real - time reverse tran- scriptase- polymerase chain reaction （RT- qPCR） was used to detect the expression of rniR -188 -5p in prostate tissues. In different prostate cell lines, miR -188 -5p expression was also detected using RT- qPCR. The miR- 188 -5p mimic was re- introduced into PC -3 and LNCaP cells by Lipfectamine 2000. The function of miR - 188 -5p on PCa cells was explored by colony formation assays, Transwell migration and invasion assays. The candidate targets for miR - 188 -5p were investigated using prediction al- gorithm provided by miRanda. Luciferase activity was measured using the dual luciferase reporter assay sys- tem. Results The expression level of miR - 188 -5p was signifieandy lower in PCa tissues than in pericarci- nous and BPH tissues （0. 996 ±0. 024, 0. 990 ±0. 094, 0. 385 ±0. 031 ; P 〈0. 05）, and miR - 188 -5p expression was comparable in pericarcinous and BPH tissues （ P 〉0. 05）. It was also found that miR - 188 -5p was significantly down - regulated in PCa cell lines as compared with that in RWPE - 1 （0. 409 ± 0. 034, 0. 358 ±0. 024, 0. 255 ±0. 018, 1. 022 ±0. 012 ; P 〈0. 01 ）. The colony formation assays showed that ec- topic miR - 188 - 5p expression significantly inhibited colony formation compared to control PC - 3 and LNCaP cells （21 ±5, 47 ±3; 25 ±4, 51 ±5; P〈0. 01）. We next found eetopic miR -188 -5p expres- sion significantly reduced the cell invasion and migration compared to control PC - 3 and LNCaP cells （ P 〈 0. 05 ）. Through computational analysis, the binding site for miR - 188 - 5p at 3 ＇ - untranslated region（3＇ -UTR） of lysosome -associated protein transmembrane 4 beta （LAPTM4B） was depicted. The lucifer- ase - based assay validated LAPTM4B was regulated by miR - 188 - 5p. Conclusion The miR - 188 - 5p was significantly down -regulated in PCa. Restoration of miR -188 -5p in PCa ceils significantly suppres- ses proliferation, migration and invasion in vitro. The miR - 188 - 5p can negatively regulate LAPTM4B expression by directly binding to its 3＇ - UTR.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2016年第12期2643-2646,共4页
Chinese Journal of Experimental Surgery
基金
武汉市科技计划项目（2015060101010049）
作者简介
通信作者：刘修恒，Email：drlxh@hotmail．com
