The Role Of MiR-133b In Prostate Cancer And Its Molecular Mechanism

This article is composed of two parts:The first part is about the function and molelular mechanism of miR-133b in prostate cancer. miRNAs are short, endogenous RNAs in cells, promoting translational repression and/or destabilization of target mRNAs, to’fine tune’ protein level in numerous biological processes. The role of miRNAs in prostate cancer remains largely unknown. microRNA-133b (miR-133b), located at chromosome 6pl2.2, has been shown to function as oncogenes or tumor suppressors in different types of cancer.At present, we investigated the role of miR-133b in prostate cancer cells. With bioinformatics and microarray profile, our lab’s previous work indicated that the expression of miR-133b increased in the presence of DHT and that miR-133b might be a candidate target directly regulated by AR. As referred to bioinformatic prediction, there were seven ARE motifs located downstream and upstream 50kb of pre-miR-133b transcription starting site. On this basis, we found that, in LNCaP cells, AR directly recruited to the seven ARE regions of miR-133b in the presence of DHT. By microarray and real time RT-PCR assay, we also observed that AR can promote the expression of miR-133b. In general, miR-133b is a target of AR.Futhermore, we investigated the function of miR-133b in various cell processes. With MTT assay, we found that overexpression of miR-133b significantly increased cell proliferation of LNCaP, and that miR-133b is essential of the basic cell survival and mediating DHT-stimulated cell activity of prostate cancer. By CCK-8 and cell cycle assay, we observed that miR-133b could potentiate cell proliferation and cycle of LNCaP and PC-3 cells; whereas using cell apoptosis and intracellular caspase assay, we confirmed that miR-133b can inhibit cell apoptosis and activated caspase-3 both in LNCaP and PC-3 cells. These results indicated that miR-133b suppressed cell apoptosis by endogenic apoptotic pathways.With microarray data analysis and bioinformatics prediction, we found RB1CC1 as a candidate target of miR-133b. By luciferase assay, we verified that miR-133b can directly recruit to the 3’UTR of RB1CC1 gene. Our data also indicated that miR-133b negatively modulates RB1CC1 expression in LNCaP cells. As a result, it is reasonable that RB1CC1 is a target of miR-133b in LNCaP cells. Next, we investigated the role of RB1CC1 in prostate cancer cells. Specially, with cell cycle and cell apoptosis assay, we indicated that RB1CC1 induced G1-S phage arrest and regulated the intrinsic apoptotic pathway and expression of important tumor suppressor genes such as RB1 and p53, etc. In addition, we explored the impact of miR-133b on genes downstream of RB1CC1 signaling and important genes involed in cell cycle and apoptotic pathways. As a result, we indicated that miR-133b enhanced the expression of p21, whereas inhibited the expression of p53, RBI and caspase-3; as referred to protein level, we found that miR-133b reduced the RB1 protein and induced the p21 protein level.Besides, we used immnuohistochemistry to assess AR and RB1CC1 protein in 135 primary prostatectomy tissue samples, and in-situ hybridization to asses the expression of miR-133b. We determined the risk of tumor recurrence in AR-positive /negative group, miR-133b-positive/negative group, and RB1CC1-positive/negative group through the Kaplan-Meier method. By immnuohistochemistry we found that it was easier for the patients with positive AR staining to experience biochemical recurrence risk (p<0.05); whereas, it was easier for the patients with negative RB1CC1 staining to experience biochemical recurrence (p<0.05). By in-situ hybridization, we found that it was easier for the patients with positive miR-133b staining to experience biochemical recurrence risk (p<0.05). By Pearson’s correlation test, a significant positive correlation was observed between AR protein and miR-133b expression (p<0.001), and a significant negative correlation was observed between AR protein and RB1CC1 protein (p<0.001).The second part is about the association between the three single nucleotide polymorphisms of steroid-5-alpha-reductase, alpha polypeptide 2(SRD5A2) gene and the risk of prostate cancer. SRD5A2 plays a crucial role in androgen metabolism pathway in human prostate. It encodes SRD5A2 enzyme, which catalyses testosterone to dihydrotestosterone (DHT). DHT is the main active structure binding with androgen receptor (AR). After the activation of AR, it further regulates a series of target genes in androgen metabolism pathway. However, no clear consensus has been reached on the association between the SRD5A2 V89L, A49T and TA repeat polymorphisms and prostate cancer (PCa) risk. Thus, we performed a meta-analysis of 31 association studies with 14726 PCa cases and 15 802 controls. We found no association between PCa and 89L compared with 89V allele [odds ratio (OR)=1.02, 95% confidence interval (CI) 0.98-1.06, pheterogeneity=0.44]. The 49T allele showed a significantly elevated effect on the high stage (Stages Ⅲ-Ⅳ) of PCa risk both under the dominant genetic model (OR= 2.13,95% CI1.44-3.15,Pheterogeneity=0.65) and in the contrast T versus,A allele (OR= 2.06,95% CI 1.41-3.02,Pheterogeneity= 0.69). There was a significantly decreased association between PCa and long TA repeat as compared versus short TA repeat (OR=0.86,95%CI0.74-1.00,Pheterogeneity=0.79). No significant between-study heterogeneity was found in all subjects under four genetic models (dominant model, recessive model, allele comparison and homozygosity comparison) for these three polymorphisms, respectively, so the fixed effects model was used to pool the result. Our result indicated that carriers of 49T might improve the risk of PCa in higher stages (Stages III-IV), carriers of long TA repeat might decrease the risk of PCa and 89L may not be an important risk factor for PCa. However, due to the limited sample sizes, this meta-analysis did not achieve sufficiently conclusive results. Still more welldesigned studies should be performed to clarify the role of these three polymorphisms in the development of PCa.