The Mechanism Of AR In The Pathogenesis Of Benign Prostatic Hyperplasia Through ATG9-mediated Autophagy

Purpose:To reveal the effect of 5?-reductase inhibitor(5-ARI)interfering with androgen receptor(AR)signaling pathway on the autophagy level of prostate stromal cells,and to explore the influence of different autophagy levels on the proliferation and apoptosis of prostate stromal cells.Furthermore,the molecular mechanism of autophagy-associated gene ATG9 on the regulation of autophagy levels in prostate stromal cells is investigatedMaterials and methods:1.Benign prostatic hyperplasia(BPH)and normal prostate transitional tissue specimens as well as clinical data in patients with BPH were collected.The expression of autophagy-related genes(LC3 and Beclin-1)and the apoptosis-related genes(cleaved caspase-3 and Bcl-2)were detected by immunohistochemistry.The relationship between autophagy and apoptotic levels in BPH tissue and clinical data was assessed.2.AR overexpressed lentiviral vector was established to stable express AR in prostate stromal cells.The expression of AR in the prostate stromal cells was detected by flow cytometry(FCM),and the expression of ARP-GFP-LC3 adenovirus was detected by Western blotting.The effect of AR signaling pathway on the autophagy level of prostate stromal cells was examined by electron microscopy.The changes of autophagy-related gene expression were detected by PCR.3.The expression of ATG9A gene in prostate stromal cells was detected by shRNA lentiviral vector to detect the changes of autophagy level of prostate stromal cells.The effect of prostate cancer on the proliferation of prostate epithelial cells and stromal cells before and after knockout of ATG9A was confirmed by nude mice subrenal capsule assay.The expression of Ki67,LC3 and ATG9 in transplanted tumor was detected by immunofluorescenceResults:1.The expression of LC3A/B in BPH stromal cells was significantly higher than that in normal prostate tissues,and the expression of Beclin-1 was significantly increased in BPH stromal cells and epithelial cells.The treatment of 5-ARI could improve the autophagy level of prostate stromal cells.2.WPMY-1-AR cells treated with lnM dihydrotestosterone(DHT)showed higher levels of basal autophagy than 10nM DHT treated group.Apoptosis of prostate cells was activated by rapamycin(RAPA),and the detection of interfering AR signaling pathway can promote the expression of ATG9A.3.We successfully constructed a stable knockdown of ATG9A expression of prostate stromal cells using shATG9A lentivirus.Western blot,RFP-GFP-LC3 adenovirus,transmission electron microscopy showed that the level of autophagy was significantly decreased after ATG9A inhibition of prostate stromal cells.Nude mice subrenal capsule assay showed that knockdown of prostate stromal cells ATG9A can significantly reduce the volume of transplanted tumor,shATG9A group LC3 scattered in the cytoplasm failed to form a positive point of the signal,immunofluorescence detection of renal tubular tumor tissue proliferation indicating that shATG9A Ki-67 positive cells decreased significantly compared to ShNC group.Conclusion:1.BPH tissue autophagy level was significantly higher than the normal prostate tissue,and long-term use of 5? reductase inhibitors can further improve the autophagy level of prostate hyperplasia stromal cells,which may lead to the progression of benign prostatic hyperplasia.2?The reason why interfere with the AR signaling pathway of prostate stromal cells can improve autophagy levels in cells is that ATG9 gene is up-regulated.3.ATG9 is involved in the regulation of autophagy levels of prostate stromal cells.Inhibition of ATG9 expression can down-regulate the level of prostate stromal cells and inhibit the progression of prostate hyperplasia.Significance:This study,for the first time,elucidates the important role of autophagy in the progression of benign prostatic hyperplasia in prostate stromal cells,suggesting that long-term administration of 5a reductase inhibitors up-regulates autophagy levels of prostate stromal cells,which may be one of the important reasons for the progress of BPH patients after drug treatment.5? reductase inhibitors combined with autophagic inhibitors can be one of the drug regimens to control BPH progression.