MicroRNA-185Directly Targets Androgen Receptor And Suppresses Proliferation, Invasion, Migration And Tumorigenicity Of Human Prostate Cancer Cells

Prostate cancer (CaP) is the most frequently diagnosed carcinoma and the secondleading cause of death among men in the Western World. Androgen receptor (AR), whichcontrols the growth and differentiation of the normal prostate, plays a pivotal role in thedevelopment and progression of CaP. AR blockage is the first-line initial treatment for CaP.The mechanisms of such fatal transition remain largely unknown. AR is expressed in bothandrogen dependent and independent CaP. Therefore, AR may play a crucial role in theprogression of androgen independence of CaP.Unfortunately, some advanced cancers will still develop into castration-resistantdisease even with maximum androgen blockade (MAB) therapy. Androgen receptor canregulate various target genes, including prostate specific antigen (PSA), which isup-regulated by androgen in the prostate and is thought to contribute to the progression ofCaP. AR and its target genes are consistently regarded as important points for revealing theprocess of castration-resistant CaP. Recent studies have shown that AR is involved in theprogression of androgen-independent CaP via several mechanisms. AR is known to beassociated with various stages of CaP progression, and therefore it is essential to know howthe expression and function of AR could be inhibited effectively so as to control theprogression of CaP. In fact, AR has been regarded as an important therapeutic target toCaP.MicroRNAs (miRNAs) are a class of endogenous non-coding small RNAs with20~25nucleotides and play important negative regulatory roles through binding to the3'untranslated regions (3' UTR) of target transcripts. MiRNAs can lead to protein translationarrest, and mRNA degradation in some cases. These effects of miRNAs would furtherinfluence the activity of downstream signal pathways. Studies have demonstrated thatmiRNAs play a role in tumorigenesis, acting as oncogenes or tumor suppressor genes.Among microRNAs, miR-185is reported to have the ability to suppress tumor growthof some cancer cell lines, such as rectal cancer, ovarian cancer and so on. However, there is no report about the effect of miR-185on the proliferation and invasion of CaP. To seewhether miR-185has correlations with CaP development, we performed a series of trial todetermine the effect of miR-185on the proliferation, migration, invasion of LNCaP cell.Additionally, we also detected whether over-expression of miR-185could inhibit tumorformation in nude mice in vivo. These trials suggest that miR-185has tumor-suppressivefunction during the development of CaP.Part â… : miR-185is down-regulated in clinical CaP tissues and miR-185directly targets androgen receptor.To compare the expression levels of miR-185in clinical CaP tissues and adjacentnon-tumorous tissues,25CaP samples and their adjacent nontumorous samples werechosen for qRT-PCR examination. The results showed that the expression level of miR-185in CaP samples was significantly lower as compared with the non-tumorous specimens. Atargets prediction tool called TargetScanHuman (www.targetscan.org) was used to searchfor potential targets of miR-185. Excitedly, the results showed that there wereconsequential pairs between miR-185and3'UTR of AR. A sequence analysis revealed thatthe putative targeting site of miR-185was located at nt173-179of the AR3'UTR.Furthermore, we performed a dual luciferase reporter assay, and the results indicated thatthe3'UTR of AR was a target of miR-185and the binding site in the3'UTR wasresponsible for the regulatory effect of miR-185on AR and its biological functions.Additionally, qRT-PCR and Western blotting analysis revealed that the expression levels ofAR mRNA and protein were down-regulated by transfection of miR-185m.Part â…¡: miR-185suppresses proliferation, invasion and migration ofLNCaP cells and induces cell cycle arrest at G0/G1phase.To investigate the effect of miR-185on cell growth, cck-8assay was performed. Theresults showed that over-expression of miR-185markedly suppressed the proliferation ofLNCaP cells. Cell cycle distributions of miR-185m and miR-NC transfectants wereanalyzed by FACS. The results showed that over-expression of miR-185resulted in asignificant decrease in the number of S-phase cells and a concomitant increase in G0/G1phase cells. Furthermore, transwell invasion assay and wound healing trial indicated thatmiR-185m transfection impaired the invasive and migratory ability of LNCaP cells. These results indicate that miR-185could negatively regulate proliferation of LNCaP cells byinhibiting cell cycle conversion, and ectopic over-expression of miR-185could effectivelyreduce the invasive and migrating activities of LNCaP cells. These data suggest thatmiR-185could be regarded as a tumor suppressor of CaP cells in vitro.Part â…¢: miR-185inhibits tumor formation and migration of LNCaP cellsin nude mice in vivo.To further determine the effect of miR-185on tumor growth in vivo,5-week-old nudemice were injected subcutaneously with LNCaP cells transfected with miR-185m ormiR-NC which were subjected to2'-ome classification. Compared with the control group,animals injected with miR-185m-LNCaP showed a significant reduction in tumor growthby weight and volume. Western blotting revealed that the expression level of AR protein inxenografts arising from injection of miR-185m-LNCaP was significantly lower than that inthe control group. Additionally, the numbers of liver nodules arising from injection ofmiR-185m-LNCaP was lower than that in miR-NC-LNCaP group. These results suggestthat miR-185could inhibit tumor growth of CaP in vivo.In summary, our findings suggest that miR-185could function as a tumor-suppressorof CaP by directly targeting AR. Down-regulation of AR by miR-185could result in cellgrowth and cell malignant characteristics of CaP. Knowing that AR plays a crucial role inthe development CaP and progression of androgen-independent transition, we think thatmiR-185can be regarded as an "inhibitor" of AR and may prove to a useful tool forsuppressing the development of initial CaP and androgen-independent tumor growth ofadvanced CaP as well.