The Role And Mechanism Of MiR Mediated By Androgen Receptor In Castration Resistant Therapy

Prostate cancer is the most frequently diagnosed cancer in developed countries with the second death rate. Many patients died as the result of distant metastases.In addition, the incidence of prostate cancer has been increasing in China. The standard treatment including suppressing production of androgen and preventing combination between androgen and androgen receptor (AR) for prostate cancer which cannot be treated by surgery has been androgen deprivation therapy (ADT) since 1940s. The reduction of AR plays an important role in prostate cancer treatment. Although it is widely used and was initially highly effective, ADT uniformly leads to the development of castration-resistant PCa (CRPC) after 18-24 months. Although such development is due to the quality and quantity of AR, the precise mechanism that governs the development of CRPC has yet to be fully realized. MicroRNAs (miR), which provide a new clue for demonstrating that development, are endogenous, single-stranded small non-coding RNAs (approximately 22-25 nucleotides) which usually cause gene silencing by reducing mRNA stability and/or translation. One kind of miR can regulate tens of mRNA. At the same time, miR is also regulated by transcription factors involved in AR. Increase or decrease of miRNAs in human CRPC could be important for progression of prostate cancer (PCa) from castration sensitive to castration resistant. It is essential for us to investigate the role of AR and miR in the development of CRPC. Furthermore, the downstream of AR and miR single pathway needs to be clarified as well.Objectives:To investigate the role of miR mediated by AR in the development of castration resistant in prostate cancer, we chose epithelial growth factor receptor (EGFR) as the downstream factor to initially explore the mechanism of such development.Methods:(1) We screened and identified the kind of miR mediated by AR regarded as transcription factor based on PROmiRNA database. (2) After collecting 24 samples of prostate cancer acquired by prostatectomy, we used RNAprep pure FFPE Total RNA Extraction Kit to extract RNA from prostate cancer FFPE. In those samples, there were 13 with AR(-) and 11 with AR(+). Then, reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time polymerase chain reaction (qRT-PCR) were applied to examine the miR which we have identified. While the expression of miR in AR(-) group was compared with AR(+) group, we compared the expression of miR in normal tissue and tumor in each group. (3) DU145 cell line was cultured for the research of mechanism due to the non-expression of AR. Lipo2000 system was applied to transiently transfect miR-inhibitor into the cell line before incubating the cells for 24-48 hours. The expression of EGFR was examined by western-blot. (4) After using the same system to transiently transfect miR-mimic into the cell line, we detect the expression of Raf, the downstream factor of EGFR, to to initially explore the miR pathway mediated by AR.Results:(1) Based on PROmiRNA database, we found the binding site of AR in the promoter region of miR-7-1 which is the precursor of miR-7. (2) Having examined the expression of miR-7 in prostate cancer FFPE, we found no significant difference between tumor and normal tissue in AR(+) (p=0.118) while the expression of miR-7 in prostate cancer was evidently less than that in normal tissue (0.002) among AR(-) group. There was no significant difference in normal tissue between AR(+) group and AR(-) group. In short, the expression of miR-7 in AR(+) was significantly more than that in AR(-). (3) After transiently transfecting miR-7-inhibitor into DU145,we found the expression of EGFR mRNA significantly increased according to the result of western-blot. (4) The mRNA and protein of raf significantly decreased in DU145 transiently transfected by miR-7-mimic based on the results of qRT-PCR and western-blot.Conclusions:During ADT, the reducing expression of AR leads to the reduction of miR-7, which can suppress the expression of EGFR. That pathway can influence the biological behavior via raf.