The Study Of Two MiRNAs And Their Targets Related To Androgen-induced AR Activating Signal System In ER-AR+Breast Cancer Cells

BackgroundAs a sex steroid hormone-responsive tumor, breast cancer has been the focus of attention of cancer researchers and its incidence and mortality rank first in female malignant tumors. Relative to estrogen receptor (ER) and progesterone receptor (PR), androgen receptor (AR) in breast cancer biological role is a neglected area in clinical and basic research. Recent years a growing number of experimental studies have shown that closely relation between AR and the breast cancer development and prognosis. Many reports showed that the AR is highly expressed in breast cancer, especially expressed in about1/2ER-negative cases and AR-positive breast cancer patients possessed better clinical course and prognosis than AR-negative cases Therefore, it is great significance to find out the mechanism of AR in breast cancer for a better understanding of breast cancer biological behavior and development of new treatment method.Previous studies show that AR is activated through androgen response element binding, thereby regulating the transcription of target genes, affecting gene expression programs within the cells, including changes in the miRNAs. In recent years, due to negative regulation for target protein expression non-coding miRNAs are the hot field of cancer research and, as a tumor suppressor gene or oncogene, miRNAs can down-regulate the expression of oncogenes or the tumor suppressor gene, respectively.Our previous study results show that the androgen—dihydrotestosterone (DHT) can inhibit the ER-AR+MDA-MB-453breast cancer cell proliferation, make cell cycle arrest in G1-S phase, and up-regulated AR protein expression, and moreover miRNA microarray hybridization results filtered out obvious up-regulated and down-regulated miRNAs. In this study, based on previous studies, two miRNAs related to AR from the screening of miRNAs were deeply studied and gained some important discoveries.Purpose1. To elucidate the correlation between let-7a and the activation of the AR and do the cell functional studies on the let-7a in order to clarify the important role among activation of the AR, let-7a and its target proteins in DHT inhibiting ER-AR+breast cancer cell proliferation.2. To explain the correlation between miR-30a and the activation of the AR, do the cell functional studies on the miR-30a, validate if the target protein of miR-30a is AR and thereby to clarify the relations between AR and miR-30a in DHT inhibiting ER-AR+breast cancer cell proliferation.Materials and methods1. MiRNA microarray hybridization results were re-analyzed and the possible miRNAs whose target gene is AR were predicted by using bioinformatic prediction software.2. By real time quantitative RT-PCR, up-regulated miRNAlet-7a and down-regulated miR-30a,b,c were validated.3. By western blot, we analyzed AR、c-Myc and k-Ras expression in breast cancer cells.4. Whether activated AR signaling pathway directly regulate let-7a or miR-30a through chromatin immunoprecipitation (ChIP) assay.5. MiRNAs were up-regulated by constructing vector and transiently transfecting with vector to breast cancer cells6. MiRNAs were down-regulated by transiently transfecting with specific antisense oligonucleotide (ASO) to breast cancer cells.7. Cell proliferation was determined by MTT assays and cell cycle was analyzed by flow cytology.8. Using dual fluorescent protein reporter assay system, it was identified that AR was target protein of miR-30a.Results1. MiRNA microarray screening results showed that a total number of94miRNA were identified using the criteria that miRNA undergo alternations at least1.5-fold. Among these miRNAs,43up-regulated miRNAs were found including let-7a, b, c, d, e, g, while51down-regulated miRNAs were identified including miR-30a, b, c. Further analysis found that in the up-regulated miRNAs only let-7a, b, c, d conformed to the criteria of more than2-fold and expression intensities differed by at least>1000.2. In all down-regulated miRNAs, miR-30a, b, c are significant because their predicted target genes include AR by Bioinformatics techniques3. Real-time quantitative RT-PCR (RQ-RT-PCR) revealed≈13-fold increase of let-7a whereas5-fold decrease of miR-30a,>2-fold decrease of miR-30b,≈2-fold decrease of miR-30c in DHT-treated cells than vehicle-treated cells. We selected up-regulated let-7a and down-regulated miR-30a to further study.4. The ChIP assay results confirmed that the activated AR directly regulates the let-7a, but does not directly regulate the miR-30a.5. Western blot results suggested that c-Myc and k-Ras were upregulated after the MDA-MB-453cells were exposed to DHT.6. RQ-RT-PCR confirmed that let-7a/miR-30a was up-ragulated by constructing vector and transiently transfecting with vector to breast cancer cells and let-7a/miR-30a was down-ragulated by transiently transfecting with specific antisense oligonucleotide to breast cancer cells.7. Cell proliferation activity increased, S-phase fraction in the cell cycle increased, the proportion of G1phase cells decreased, and let-7a known target proteins—c-Myc and k-Ras were upregulated in the let-7a low expression group because MDA-MB-453cells were transfected by let-7aASO. In let-7a high expression group because MDA-MB-453cells were transfected by vector, the results were opposite.8. Cell proliferation activity increased, S-phase fraction in the cell cycle increased, the proportion of G1phase cells decreased and miR-30a predicted target proteins—AR were upregulated in the miR-30a low expression group because MDA-MB-453cells were transfected by miR-30aASO. In miR-30a high expression group because MDA-MB-453cells were transfected by vector, the results were opposite.9. Dual fluorescent protein reporter assay system results showed that AR was target protein of miR-30a.Conclusion 1. As a transcription factor, activated AR act on the promoter of let-7a and directly up-regulated let-7a.2. AR was the target protein of miR-30a and was directly regulated by miR-30a.3. Both let-7a and miR-30a were cancer suppressor miRNAs.4. Androgen-induced AR up-regulation of let-7a that target k-Ras and c-Myc may play a key role in the androgen inhibition of ER-, PR-, AR+MDA-MB-453breast cancer cell proliferation.5. miR-30a is a tumor suppressor miRNA and androgen-induced AR can down-regulate miR-30a, but do not act on the promoter of miR-30a. As a androgen responsive miRNA, miR-30a may play a role in the androgen inhibition of ER-, AR+MDA-MB-453breast cancer cell proliferation mainly through it down-regulating its target protein—AR.