MiR-185Target-regulates Androgen Receptor Expression In Prostate Carcinoma Cell Line LNCaP

Androgen receptor (AR) is a ligand-dependent transcription factor, which plays the most significant role in Prostate cancer (PCa) from disease initiation to disease progression and the development of treatment. Early androgen-dependent prostate tumors is androgen-dependent, blocking the androgen is effective in the treatment of early prostate cancer. But this treatment can cause cancer cells to androgen-independent growth. Used to think that the lack of AR protein in androgen-independent prostate cancer, but now the study found that in androgen-dependent prostate cancer the expression level of AR and AR-positive cells is not significantly reduced, even the over-expression. In view of this, block the expression of AR, inhibiting its functional activity are effective treatment targeted both in androgen-dependent and androgen-independent prostate cancer.New study finds that miRNA play an important role in the pathology of tumors in a variety of diseases in humans. miRNA can be as a tumor suppressor gene or suppressor gene. According to the study, miR-185play as a suppressor gene in a variety of tumor, inhibiting of lung cancer, breast cancer, ovarian cancer and colorectal cancer. miR-185may be through different mechanisms of inhibiting of cell proliferation in different tumor cells. Our research found that miR-185also inhibits proliferation of prostate cancer cell line LNCaP. For further discussion on the mechanism of miR-185inhibiting proliferation of LNCaP, we found the AR is the target gene of miR-185by miRDB database query. To better understand the miR-185 down-regulates androgen receptor expression in prostate carcinoma cell line LNCaP: The relation between expression of the miR-185and status of cells were investigated by cell apoptosis and viability assay. Real-time quantitative PCR (RT-qPCR) analyses were performed to measure the AR mRNA and the miR-185levels in prostate cancer cell line LNCaP. Western blotting were used to compare AR protein expression in the different cells. Dual luciferase assays serve as molecular mechanisms between miR-185and AR in cells.The results show that AR is a direct target gene of the miR-185. In addition, the has-miR-185inhibited activity of the AR mRNA by binding the target site in the AR-3’UTR, markedly reducing the AR protein levels, and down regulating the expression of AR target genes by AR signaling axis, and inhibit of cell proliferation and increase of cell apoptosis. Our results suggest that the miR-185is important in prostate carcinogenesis, and affords a new therapeutic option for the progression PCa.Part1:Down-regulatory effect of miR-185on AR activity in prostate cancer LNCaP cellsObjective:To evaluate the down-regulatory effect of miR-185on AR activity in prostate cancer LNCaP cells.Methods:1. LNCaP cells were seeded into6-well plates and transfected with either miR-185mimic or inhibitor or NC. Total RNA was extracted48h after transfection using Trizol reagent. Reverse transcription and quantitative Real-time was carried out for miR-185and the AR, U6and GAPDH was used as a control gene for normalization. The relative amount of the miR-185was determined by2-ΔΔCT methods.2. LNCaP cells were transfected with indicated amounts of miR-185mimic or inhibitor for48h and72h. Western blot were carried out to detect the effects of miR-185mimic or inhibitor on the AR expression. Beta-actin expression was used as a loading control in Western blot. 3. The wild type (WT)3’UTR of the human AR gene was amplified from human AR cDNA by using PCR method, and cloned into pMIR-REPORT Luciferase, which is named pMIR-AR-3’UTRw. PCR primers used to amplify the AR-3’UTR are ARF and ARR. The deletion mutagenesis of the putative target site for miR-185in WT-3’UTR of AR was carried out by using two-step PCR method. Using pMIR-AR-3’UTRw as the template,5’flanking fragment of the deletion region was PCR amplified with the primers, ARF and DR, and3’flanking fragment was PCR amplified with the primers, ARR and DF. The two fragments were linked by PCR ligation with the primers, ARF and ARR. The ligated fragment was cloned into the pMIR-REPORT Luciferase, which is named pMIR-AR-3’UTRm.4. LNCaP cells were transfected with either miR-185mimic or inhibitor for48h, then cotransfected with either pMIR-AR-3’UTRw or pMIR-AR-3’UTRm for another48h. Luciferase activity was detected using Dual Luciferase Assay System and was plotted as ratio of firefly to renilla luciferase activity.Results:1. Using Quantitative Real-time PCR technology, we measured the expression of the miR-185in the treated LNCaP cells. The date showed that all kinds of miRNA are highly expressed in the treated prostate cancer LNCaP cells2. We detected the AR expression by Western blotting. The results showed that the transfection of miR-185mimic for72h markedly reduced the AR protein levels in LNCaP cells; while the transfection of miR-185inhibitor increased the AR protein levels in LNCaP cells.3. Each of these constructs was cotransfected to LNCaP cells with either miR-185mimic or inhibitor. Luciferase activity was measured48h post transfection. The results showed that miR-185mimic obviously reduced, while miR-185inhibitor increased the luciferase activity of the pMIR-AR-3’UTRw. However, neither miR-185mimic nor inhibitor changed luciferase activities of pMIR-AR-3’UTRm or pMIR-Report plasmid.Conclusion: miR-185could reduce the AR protein levels by binding the predicted target site in the AR-3’UTR, but not by causing AR mRNA degradation.Part2:Down-regulatory effect of miR-185on ARE activityObjective:To evaluate the down-regulatory effect of miR-185on ARE activity in prostate cancer LNCaP cells.Methods:1. For the construction of pGL4-ARE reporter plasmid, oligonucleotides of the putative ARE were synthesized. The double-stranded ARE was generated by annealing equal amounts of sense and antisense. Then it was inserted to upstream of the TATA box in the pGL4.23[luc2/minp] vector to generate the recombinant plasmid of ARE-TATA box-luciferase reporter, named as pGL4-ARE. Nucleotide sequences of the pGL4-ARE were confirmed by DNA sequencing.2. LNCaP cells were seeded in24-well plates and transfected for24h with either miR-185mimic or inhibitor or NC using siPORTTM NeoFXTM Transfection Agent, then these LNCaP cells were again cotransfected with pGL4-ARE or pGL4.23[luc2/minp] by using FuGENE HD for48h. Firefly (M1) and Renilla (M2) luciferase activities were measured by using Dual Luciferase Assay System according to the manufacturer’s instructions. Transfection efficiency was normalized by the control vector pGL4-TK.3. LNCaP cells were transfected with either miR-185mimic or inhibitor, and the cells were harvested72h post transfection. Total RNA was extracted and qRT-PCR was performed to detect the effects of miR-185on the expression of NKX3.1and PSA genes. GAPDH was used as a control for normalization, and the relative expression level of NKX3.1and PSA mRNA was relative to GAPDH mRNA. The data is the means of three individual values±S.D.Results:1. We synthesized ARE sequence and inserted it to upstream of TATA box in pGL4.23[luc2/minp] plasmid, named pGL4-ARE. Nucleotide sequences of the pGL4-ARE were confirmed by DNA sequencing. 2. pGL4-ARE was cotransfected to LNCaP cells with either miR-185mimic or inhibitor. Then results measured with luciferase activity showed that the miR-185reduced luciferase activity of the pGL4-ARE by about40.5%as compared to that with the NC mimic; has-miR-185inhibitor increased luciferase activity of the pGL4-ARE by about30.8%as compared to that with the NC inhibitor. These data demonstrate that miR-185substantially inhibit ARE activity.3. Using Quantitative Real-time PCR technology, we measured the expression of the NKX3.1and PSA mRNA in the treated LNCaP cells.The results of qRT-PCR showed that the expression of NKX3.1and PSA mRNA in LNCaP cells transfected with miR-185mimic was reduced compared with that in the cells transfected with NC mimic, while the expression of NKX3.1and PSA mRNA in LNCaP cells transfected with miR-185inhibitor was increased compared with that in the cells transfected with NC inhibitor.Conclusion:miR-185could reduce the expression of AR target genes by inhibiting the androgen responsive element activity.Part3:The effect of miR-185on the apoptosis and proliferation of prostate cancer LNCaP cellsObjective:To evaluate the effect of miR-185on the apoptosis and proliferation of prostate cancer LNCaP cellsMethods:1. LNCaP and PC-3cells were transfected with either miR-185mimic or inhibitor. Cell viability was detected24-96h post transfection using MTT method. Data were shown as the mean of six individual values±S.D.2. LNCaP cells were transfected with either miR-185mimic or inhibitor for72h,96h and96h, and apoptosis was assessed using Hoechest33258.Results: 1. The results by the MTT method showed that miR-185mimic reduced by29.8%, while miR-185inhibitor increased by15.9%of viable LNCaP cells. The same transfection in PC-3cells no significant effects of miR-185mimic or inhibitor on the cell proliferation.2. The results by the Hoechest33258showed that after the tansfection of miR-185mimic, much more LNCaP cells displayed chromatin condensation and marginalization compared with the NC mimic transfected cells. In contrast, the transfection of miR-185inhibitor resulted in decreased number of LNCaP cells with nucleic chromatin condensation compared with the transfection of NC inhibitor.Conclusion:The effect of miR-185on cell growth is related to AR down-regulation and miR-185induced cell growth inhibition is mediated mainly by AR repression.