PTEN Is Regulated By Multiplex MicrorRANs In Prostate Cancer Cells

As one of the most common malignant tumors in men, the morbidity and mortality of prostate cancer significantly increased in our country in the recent years. However, the mechanism of prostate cancer pathogenesis is not well elucidated. PTEN, a tumor suppressor gene, has been found to play vital roles in prostate carcinogenesis and progression, which is often inactivated by mutation, exonic deletion, and loss of transcript in advanced prostate cancer. As one of the main post-transcriptional regulation mechanisms of gene expression, microRNA (miRNA) is known to be closely associated with carcinogenesis and tumor progression in various human cancers, including prostate cancer. This thesis focused on exploring the molecular mechanisms of PTEN expression regulation by miRNAs in prostate cancer cells, which will help to understand the molecular mechanisms of prostate carcinogenesis and progression, even to give some clues for the diagnosis, gene therapy and/or prognosis of prostate cancer.In our study, we first investigated the candidate miRNAs of PTEN by by TargetScan Human Prediction and Dual-Luciferase Reporter Aassays.We found that there exists multiple targets of several miRNAs in the 3’UTR of PTEN mRNA, those are miR-19b, miR-23b, miR-26a and miR-92a, which distribute in several different locus in the 3’UTR and show different "seed" regions matches between miRNA and 3’UTR. These four miRNAs could regulate the reporter gene expression at the post-transcriptional level by interaction with PTEN mRNA 3’UTR. Then we selected PNT1B, a normal human prostate epithelial cell line, and DU145, a prostate tumor cell line for further investigations after we detected the endogenous expression of PTEN and miR-19b、miR-23b、miR-26a、miR-92a in several normal prostate cell lines and prostate cancer cell lines, including human prostate epithelial cell line PNT1B, Benign prostatic hyperplasia epithelial cell line BPH-1, and human prostate cancer cell line 22RV1, DU145, LNCaP and PC-3. After overexpression of one, or two or three miRNA(s) in PNT1B and DU145 by knock-in the miRNA-expression plasmids, we detected PTEN expression level by real-time RT-PCR assay Western blot analysis, and found that the overexpressed miR-19b, miR-23b, miR-26a and/or miR-92a could effectively repress the PTEN expression in prostate epithelial cells or cancer cells. Thereafter, we knock-out these miRNAs by transfection of the anti-miRNAs, those are anti-miR-19b, anti-miR-23b, anti-miR-26a and/or anti-miR-92a, and we found that PTEN expression level in prostate epithelial cells or cancer cells was went up by antisense neutralization of the corresponding miRNAs. The results of these knock-in and knocko-out experiments might prove that miR-19b, miR-23b, miR-26a, and miR-92a jointly participated in the regulation of PTEN expression, and miR-26a, and miR-92a might be the key regulators among these four miRNAs. Furthermore, we knocked-in or knocked-out these miRNAs according to the previous methods, and found that miR-19b, miR-23b, miR-26a, and miR-92a could singly or jointly targeted regulate and repress the key signal molecules of PI3K/Akt pathway, including PIK3CA (p110α), PIK3CD (p110δ), PIK3R1 (p85) and Akt, in the meanwhile. These results might reveal the complexity of PTEN expression regulation by miRNAs, that is to say, there existed the neutralization effect from the antagonistic action and the synergistic effect from upstream gene regulation. We then explored whether the prostate epithelial cells or cancer cells could proliferate or not by overexpression of these four miRNAs, and observed that these four miRNAs could promote the cell proliferation of the prostate epithelial cells or cancer cells, especially miR-26a and miR-92a could significantly expand the cells, which also suggested that miR-26a and miR-92a might act as a oncomiR in prostate cancer.Taken together, our results indicated that miR-19b, miR-23b, miR-26a, and miR-92a could jointly regulate PTEN expression at the post-transcriptional level, and further targeted regulate the key signal molecules of PI3K/Akt pathway, including PIK3CA (p110α), PIK3CD (p110δ), PIK3R1 (p85) and Akt, hence promote the prostate epithelial cell or cancer cell proliferation. Given these results, our findings will help to elucidate the molecular mechanisms of PTEN expression regulation by miRNAs, and even give new clues to PTEN gene therapy of prostate cancer.