Study On The Function Of MiR-200b-3p To The Proliferation Of Androgen-independent Prostate Cancer Cells And The Molecular Mechanism

Prostate cancer is one of the common malignant tumors of the male genitourinary system and accounts for the second most common cause of death in males. Prostate cancer cell growth is dependent on the presence of androgens. Androgen hormone-blocking treatments (castration therapy) of prostate cancer, can inhibit tumor growth, and improve disease-free survival for the majority of patients by1.5to3years. However, the tumors tend to relapse following this disease free period with androgen independent proliferation ability. This kind of prostate cancer is named as androgen-independent prostate cancer (AIPC) or castration resistant prostate cancer (CRPC). For these AIPC cases, the current treatment is limited and the prognosis is poor.Over the years, the research on AIPC mechanisms focuses on the androgen receptor (androgen receptor, AR). However, the mechanism of AIPC development remains unclear. Such mechanisms are further complicated with the recent discovery of microRNA (miRNA), a class of non-coding RNAs that regulate gene expression at the post-transcriptional level. Accumulating evidence indicates that microRNAs play critical roles in multiple biological processes, including cell cycle control, cell growth and differentiation, apoptosis, and embryological development. Some miRNAs involved in the occurrence of AIPC have been reported. White et al., found a small part of miRNAs abnormally expressed in AIPC cell lines. Sung et al., found a significantly high expression of miR-221/222in AIPC cell lines relative to the ADPC cell lines, suggesting its role in the occurrence and development of AIPC. Ma et al., found that miR-616is overexpressed in malignant prostate tissue. Over expression of miR-616in LNCaP cells (androgen dependent prostate cancer, ADPC) in vitro, can promote cell proliferation in a non-androgen environment.Studies have shown that one miRNA regulates more than one target gene, and one gene can be regulated by more than one miRNAs. Thus, there remain many questions. Are there any other miRNAs invoved in AIPC regulation?What function of these miRNAs in AIPC progress?How these miRNAs regulate AIPC progress?Can these miRNAs be candidate treatment target of AIPC?In order to reveal the role of miRNAs played in AIPC, We conducted the following experiments.First, the dependce of prostate cancer cell lines LnCap and PC3was verified.Second, the differential miRNAs and cancer related genes were screened by Quantitative real-time PCR (Q-PCR).Third, the correlation between miRNAs and genes was analysed by literature search and bioinformatics method.Fourth, the expression level of differential miRNA and gene was verified. The expression of interested miRNA regulated by the specific gene and the role of this regulation played in AIPC proliferation were invested.Fifth, the proteins regulated by the interested miRNA were identified by proteomics method, and the molecular mechanism of the proliferation regulation by interested miRNA was explored.We compared several relevant miRNAs and cancer related genes between the ADPC cell line (LnCap) and the AIPC cell line (PC3) using Q-PCR and western-blot, and found p73and miR-200b-3p were co-downregulated in the PC3cell line. The present study will investigate the regulation between miR-200b and p73, and whose role in the androgen-independence of prostate cancer cells.We first screened several miRNAs by Q-PCR. Among the differential microRNAs, miR-200b-3p was significantly repressed in PC3-Ad. This is consistent with the findings of Xu et al. Several studies have shown the function of miR-200b in cancer. Kong et al. reported that miR-200b regulates PDGF-D-mediated epithelial-mesenchymal transition, adhesion, and invasion of prostate cancer cells. However, the function of miR-200b-3p in AIPC is riot clear. Our results indicated that down-regulation of miR-200b-3p increased the proliferation of ADPC cells cultured with androgen-free medium, while up-regulation of miR-200b-3p decreased the proliferation of AIPC cells. Thus, low expression of miR-200b-3p may contribute to the androgen-independent growth of prostate cancer.We detected several proteins which correlated with cancer, and found p73expression showing a positive correlation to miR-200b-3p. p73participates in the apoptotic response to DNA damage, and may be a tumor suppressor protein. There is also some evidence indicating p73expression decreases in AIPC. We further detected the effect of p73expression changes on the proliferation of prostate cance cells cultured with androgen-free medium. Our results showed that inhibition of p73expression promoted prostate cancer cell proliferation without androgen, while over-expression of p73inhibited prostate cancer cell proliferation without androgen. Thus, p73inhibited androgen-independent prostate cancer growth.Our results demonstrated that the expression and function of p73strongly correlated to miR-200b-3p. We wanted to clarify the relationship between p73and miR-200b-3p in AIPC. p73was over-expressed in the PC3-Ad cell subline, while miR-200b-3p expression increased accordingly. While p73was inhibited in LnCap-Ad cell subline, miR-200b-3p expression also decreased significantly. The regulation of miR-200b-3p by p73is also supported by Emily C et al. for other cancers. They have proved that p73directly regulates miR-200transcription by chromatin immunoprecipitations method.The regulation of p73by miR-200b-3p in AIPC cells was also explored because the bioinformatic resource TargetScan (http://www.targetscan.org/) predicted TP73may be a potential miR-200b-3p target. Our results indicated that neither over-expression nor inhibition of miR-200b-3p affected the expression of p73in prostate cancer cell lines with androgen-free culture medium.Next, we identified the proteins regulated by miR-200b-3p by proteomics. The result showed14differential expressed proteins. Among the proteins,5up regulated in miR-200b-3p over-expressed PC3cells and9down regulated. The former includes: Stathmin (STMN1), POTE ankyrin domain family member E (POTEE), Inosine triphosphate pyrophosphatase (ITPA), UV excision repair protein RAD23homolog B (RAD23B), Importin subunit alpha-4(KPNA4). The latter includes:Thioredoxin (TXN), Cytochrome c oxidase subunit5A, mitochondrial (COX5A), Peroxiredoxin-2(PRDX2), Protein Drl (DR1), polypeptide-associated complex subunit alpha (NACA), Small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA), Elongation factor1-delta (EEF1D), Nucleophosmin (NPM1), Vimentin (VIM).PRDX2was identified as a target gene of miR-200b-3p by TargetScan, http://www.targetscan.org/. The expression of Peroxiredoxin-2and another protein Drl in the cells miR-200b-3p over-expressed or not were verified by Q-PCR and western-blot. The results were accordance with which of proteomics.Peroxiredoxin-2plays bifunction in cancer cells. This protein may have a proliferative effect and play a role in cancer development or progression. But this protein can prevent cancer cell transformation from normal cell. Thus, we speculated that peroxiredoxin-2inhibited by miR-200b-3p, is one of the mechanism of miR-200b-3p’s anti-cancer function.The proteins interaction among the proteins identified by proteomics was analysed by informatics STRING (http://string-db.org/). The result indicated that most proteins down-regulated in the PC3cells with miR-200b-3p over-expression were connected by ubiquitin, including PRDX2、TNX、VIM、EEF1D、SGTA、NACA、 NPM1. Almost al of these proteins directly or indirectly can promote the cell proliferation. Furthermore, we found some ubiquitin-specific proteases (USPs) may be the target gene of miR-200b-3p by Targetscan. Thus, we deduced that miR-200b-3p can inhibit the expression of USPs, then ubiquitin pathway increased, many proteins including Thioredoxin, Vimentin, Elongation factor1-delta, Small glutamine-rich tetratricopeptide repeat-containing protein alpha, Nucleophosmin were degraded by ubiquitin, the AIPC cells proliferation was inhibited. In sum up, we concluded:1. miR-200b-3p were regulated by p73in AIPC. miR-200b-3p was increased by p73over-expression.2. miR-200b-3p can inhibit the expression of Peroxiredoxin-2, then inhibited the proliferation of AIPC.3. miR-200b-3p can inhibit the expression of some USPs including ubiquitin specific peptidase27, X-linked; ubiquitin specific peptidase25; ubiquitin specificpeptidase31; ubiquitin specific peptidase46; ubiquitin specific peptidase47.4. miR-200b-3p can increase the expression of UV excision repair protein RAD23homolog B.5. The USPs inhibition and RAD23homolog B elevation increased the ubiquitination of some proteins including Thioredoxin, Vimentin, Elongation factor1-delta, Small glutamine-rich tetratricopeptide repeat-containing protein alpha, Nucleophosmin, then the proliferation of AIPC cells wes inhibited.