Analysis Of Up-regulation Of PDLIM4Expression In Drug-Resistant Prostate Cancer

Prostate cancer (prostate cancer, PCa) has become a severe threaten to human health in the old men, and also in China there are much more advanced patients than European countries as the average lifespan increasing along with changes in living environment and diet habits. PCa is an androgen-dependent tumor, whose proliferation is mainly mediated by androgen/androgen receptor (AR) signal pathway, so androgen ablation serves as a standard treatment for advanced patients. But eventually this kind of PCa will produce hormone resistance, then progress to hormone refractory prostate cancer (HRPC), and finally approximately more than half of the patients have bone or soft tissue metastases with high mortality. Compared with other clinical drugs such as mitoxantrone, docetaxel is a standard drug for HRPC patients, however, about50%of patients have no response to docetaxel, and have significantly toxic response, then HRPC become more malignant and drug resistance because of intrinsic and acquired mechanisms. In prostate cancer, different changes can happen during the process of drug resistance. Among them, abnormal expressions of oncogenes and tumor suppressor genes can take part in drug-resistant process. We focus on PDLIM4gene, also called RIL (reversion-induced LIM gene), maps to chromosome5q31.1, and is identified as a candidate tumor suppressor without well-known functions. In prostate cancer, hypermethylation of PDLIM4promoter leads to its loss of expression, of which the mechanism is not clear.Epigenetic regulation generally includes DNA methylation, histone modifications, chromatin remodeling and RNA interference (e.g. microRNAs) etc. And DNA methylation is one of the most common epigenetic regulatory mechanisms without changes in DNA sequences, which is essential for the correct development of the organisms. The DNA methylation is mainly catalyzed by three enzymes called DNA methyltransferases (DNMT1, DNMT3A, DNMT3B) which introduce the methylation group from a molecule of S-adenosylmethionine. Methylation in transcription regulatory regions can result in the inhibited expression of genes, and silence of tumor suppressor genes can lead to pathogenesis and progression of tumors. Methylation of DNA is a reversible change, and gene can reexpress by5-Aza-2’-deoxycytidine (5-Aza), which is a potent DNA methyltransferase inhibitor.Compared with androgen-independent PC3cells in gene expression profiles, docetaxel-resistant cells PC3/Doc has higher PDLIM4expression firstly, so the functions of PDLIM4in PC3/Doc were preliminarily investigated, whether PDLIM4could be a new target for treating HRPC.Objective:To study the regulation of PDLIM4expression in prostate cancer, meanwhile investigate its function in PC3/Doc cells.Methods:1. Different cells were used to identify PDLIM4expression by qRT-PCR, Western blot, Immunofluorescence technique (IF).2. The activities of DNMTs were also tested in PC3and PC3/Doc.3. The methylation levels of PDLIM4promoter in different cells were tested by mehtylation-specific PCR (MSP).4. RNA interference (RNAi) was used to investigate the function of PDLIM4in PC3/Doc cells.5. Transfection of plasmid MBD2was used to find the regulation of PDLIM4.Results:’. High expression of PDLIM4in drug-resistant cells is tissue-specific.1. PDLIM4was overexpressed in RWPE1cells, not expressed in DU145or LNCaP cells, slightly detected in PC3cells but predominantly evidenced in its multidrug resistant PC3/Doc cells (P<0.05) 2. K562, K562/A02can also express PDLIM4, but lower in K562/A02. H46(h H460/Doc have NO PDLIM4expression.二. DNA methylation is not the only factor which can affect PDLIM4expression.1. DNMTs has lower expression in RWPE1.2.5-Aza can obviously restore PDLIM4expression in LNCaP cells, but has lower effect in PC3cells.3. DNMTs have lower activities in PC3/Doc than PC3although PC3/Doc and PC3have similar DNMTs expression.4. Methylation level of PDLIM4promoter in PC3/Doc is lower than PC3.5. PC3/Doc has no MBD2expression, overexpression of MBD2has no effect on PDLIM4expression.6. TSA has no effect on PDLIM4expression.三. Knockdown of PDLIM4can decrease docetaxel sensitivity.Conclusion:High expression of PDLIM4in drug-resistant cells is tissue-specific. Other mechanisms can take part in PDLIM4regulation in PC3except DNA methylation, further investigations should be done in order to know the exact functions of PDLIM4gene.