摘要
目的验证丝裂原活化蛋白激酶1（MAPK1）是否为前列腺癌细胞中微小RNA（miRNA，miR）-129的靶基因，并探讨其分子调控机制。方法应用生物信息学软件预测并筛选靶基因；构建靶基因3’端非编码区域（3’-UTR）双荧光素酶野生型载体及结合位点突变型载体，分别与miR-129模拟物片段（has-miR-129mimic）和无关序列（scramble）对照共转染前列腺癌PC-3细胞，48h后行双荧光素酶报告基因检测；实时荧光定量聚合酶链反应（Real-timePCR）检测转染48h后PC-3细胞中靶基因的mRNA表达；Westernblot检测靶基因及其下游调控基因的蛋白表达。结果筛选MAPK1为miR-129可能的靶基因；过表达miR-129后，野生型pMIR—MAPK1-WT的荧光素酶活性值（0．495±0．070）明显低于scramble对照组（1．000±0．128，P〈0．01）。突变型各组与对照组间比较差异均无统计学意义（P〉0．05）；过表达miR-129后MAPK1mRNA相对表达量（0．720±0．041）低于scramble对照组（1．000±0．056，P〈0．01）；过表达miR-129后MAPK1、磷酸化转录信号转导子与激活子3（P—STAT3）与bcl-xL的蛋白表达量下降（P〈0．01），总STAT3蛋白表达量无明显变化，差异无统计学意义（P〉0．05）。结论MAPK1可能是miR-129直接作用的靶基因，前列腺癌细胞中miR-129可通过抑制MAPK1信号通路的活性发挥抑癌作用。
Objective To verify whether mitogen -activated protein kinase 1 （MAPK1） is the target gene of microRNA （miR） - 129 in prostate cancer cells, and to explore the corresponding molecular mechanism. Methods Target genes of miR - 129 were predicted and screened by bioinformatics software. A target gene 3＇ untranslated region（3＇ -UTR） dual luciferase wild- type vector and binding site mutant vectors were constructed, which were co- transfected respectively with the has- miR- 129 mimic and scramble mimic into PC - 3 cells, and 48 h later the luciferase activities were detected. The mRNA expres- sion levels of target gene were detected by real - time fluorescence quantitative polymerase chain reaction （ Real-time PCR） after mimic transfection into PC - 3 cells at 48 h. The protein expression levels of target gene and its downstream regulation genes were detected by Western blotting. Results MAPK1 was screened as a possible target gene of miR -129. After miR- 129 over- expression, the luciferase activity values of pMIR_MAPKI_WT （0. 495 _± 0. 070） were significantly lower than those of scramble control groups （ 1. 000 ± 0. 128 ,P 〈 0. 01 ）. The differences between the mutant groups and control groups had no statistical significance （P 〉0. 05）. The mRNA relative expression levels of MAPK1 （0. 720±0. 041 ） af- ter miR - 129 over - expression was lower than those in scramble control groups （ 1. 000 _± 0. 056, P 〈 0. 01 ）. The protein expression levels of MAPK1, phosphorylated signal transducers and activators of tran- scription 3 （ p - STAT3） and bcl - xL were decreased （ P 〈 0. 01 ）, and the total STAT3 protein expression levels had no significant change （P 〉 0. 05 ）. Conclusion MAPK1 may be a direct target gene of miR - 129. MiR - 129 may play anti - tumor roles in prostate cancer cells through inhibiting the activities of MAPK1 signaling pathway.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2016年第6期1501-1503,共3页
Chinese Journal of Experimental Surgery
