Study Of The MiR-194-1 Promoter Activity And Migration And Invasion In Prostate Cancer Cells Influenced By Hypoxia

This study was to investigate the effect of hypoxia on the activity of miR-194-1 promoter,the migration and invasion ability of DU-145 cells,as well as to study the relationship between HIF-1? and miR-194-1.The purpose of the experiment is to reveal the role and significance of HIF-1? and mi R-194 in the pathogenesis of prostate cancer,which make it possible to the progression,prognosis and pathologic diagnosis of prostate cancer,as well as the mi R-194 and HIF-1? as the target gene in the therapy of prostate cancer in the future.Firstly,the promoter sequence and hypoxia response element(HRE)of miRNA-194-1 were predicted based on the epigenetic database.Secondly,we constructed the luciferase reporter vector containing the promoter sequence and the truncated expression vector.After that,these vectors were transiently transfected into the 293 T cell to determine the promoter sequence of miR-194-1.Thirdly,the promoter vector deleted the HRE sequence was transfected to cell and examined the promoter fluorescent activity in order to explore whether HRE is contained in the core promoter sequence.And then,the effect of hypoxia on the expression of HIF-1? and miR-194-1 in prostatic epithelial cells and prostate cancer cells was also studied.To further validated the relationship between hypoxia and miR-194-1,we built HIF-1? overexpression vector and synthesized HIF-1? siRNA and then detected the expression level of HIF-1? and miR-194-1 through the RT-qPCR and Western Blot experiments.Lastly,the effects of hypoxia on the migration and invasion of prostate cancer cell DU-145 were investigated by scratch test and transwell test at the cellular level.RESULTS:(1)The pGL-3-Basic-194-1 and pGL-3-Basic-194-1-? HRE luciferase vector was successfully constructed and then transfected into the 293 T cell to detect the fluorescent activity.The fluorescent activity of pGL-3-Basic-194-1 promoter was significantly increased after transfection.However,the fluorescent activity of pGL-3-Basic-194-1-HRE was lower than that of empty vector,and the fluorescent activity did not change under normoxic and anoxic conditions,indicating that the activity of promoter was significant reduced after deletion of HRE,and no longer regulated by hypoxia.It was confirmed that the HRE sequence was included in the mi R-194-1 core promoter sequence.(2)The expression levels of miR-194-1 in prostate cancer cells were lower than that of prostate epithelial cell lines,suggesting that it could be used to diagnose and treat of prostate cancer in future.(3)HIF-1? siRNA and overexpression vector were transfected into the 293 T,and detected that the overexpression of HIF-1? may down-regulate the expression of miR-194-1,while inhibiting of HIF-l? results in up-regulation of miR-194-1.(4)Overexpression of HIF-1? or hypoxia enhanced the migration and invasion of androgen-independent prostate cancer DU-145 cells.However,siRNA inhibited the expression of HIF-1?,and decresed the cell migration and invasion ability.In summary,we constructed miR-194-1 promoter vector and verified that hypoxia regulated the transcriptional regulation of miR-194-1 promoter.In addition,hypoxia or HIF-1? overexpression could promote the migration and invasion of DU-145 cells,while HIF-l? siRNA inhibited DU-145 migration and invasion.