The Potential Value And Mechanism Of Cir-ITCH In The Treatment Of Castration Resistant Prostate Cancer

Prostate cancer is the fastest growing male genitourinary system tumor in China in recent years.Androgen deprivation therapy is the standard treatment for advanced prostate cancer.While almost all the patients progress to castration resistance stage after a period of treatment.It is more difficult to treat castration resistance prostate cancer.The mechanism of castration resistance remains to be further studied.In addition to androgen receptor pathway,there are evidences that activation of Wnt/β-catenin pathway and PI3K/AKT/m TOR pathway are related to the development of castration resistance.Inhibitors of these two pathways are considered to have the potential to treat castration resistant prostate cancer.Circular RNA is a class of RNA molecules that form a ring structure by covalent bonds.It is widely involved in physiological and pathological processes and plays an important role in the oncogenesis and progression of tumors.Cir-ITCH is a widely studied circular RNA,which plays an important role in the inhibition of colon cancer,lung cancer,esophageal cancer and bladder cancer.These results showed that cir-ITCH can inhibit Wnt/β-catenin pathway through sponge different mi RNAs.However,the role of cir-ITCH in prostate cancer remains to be studied.Therefore,present study aims to explore the role of cir-ITCH in prostate cancer,especially in castration resistant prostate cancer.This research falls into three parts: Part Ⅰ: Detection of the expression of cir-ITCH in prostate cancer tissues,paracancerous tissues,prostate cancer cells and normal prostate epithelial cells.Part Ⅱ: Verification of the interaction between cir-ITCH and mi R-17 by in vitro experiments.Part Ⅲ: Observation of the effects of overexpression of cir-ITCH and mi RNA-17 on the proliferation,migration and invasion of androgen dependent prostate cancer cells(LNCa P)and castration resistant prostate cancer cells(PC-3).To explore the possible mechanism involved,western blot was used to detect the expression levels of the representative proteins of Wnt/β-catenin and PI3K/AKT/m TOR pathway in prostate cancer cells transfected cir-ITCH,and the effect of exogenous androgen or androgen receptor antagonist on the expression.Part one Expression of circular RNA cir-ITCH in prostate cancer tissues and cellsObjective: To detect the expression of cir-ITCH in prostate cancer and paired paracancerous tissue samples,as well as in two kinds of prostate cancer cells(LNCa P and PC-3)and normal prostate epithelial cells(RWPE-1).Methods: Quantitative reverse transcription polymerase chain reaction was used to detect the expression of cir-ITCH in 10 pairs of prostate cancer tissues and paracancerous tissues which were obtained from the prostatectomies performed between January and December 2018,as well as in three types of cells including androgen dependent prostate cancer cells(LNCa P),castration resistant prostate cancer cells(PC-3)and normal prostate epithelial cells(RWPE-1).Results: The relative expression of cir-ITCH was 4.04 ± 0.22 in paracancerous tissues and 1.28 ± 0.06 in prostate cancer tissues,respectively.The expression of cir-ITCH was significantly decreased in prostate cancer tissues(P < 0.001).The relative expression levels of cir-ITCH in RWPE-1cells,LNCa P cells and PC-3 cells were 1.0 ± 0.04,0.52 ± 0.04 and 0.48 ± 0.03,respectively.The expression levels in prostate cancer cells were significantly lower than that in normal prostate epithelial cells(P < 0.05),and there was no significant difference between the two kinds of prostate cancer cells(ns,no significance).Summary: The expression of cir-ITCH is significantly decreased in prostate cancer tissues and cells.There is no significant difference in cir-ITCH expression level between androgen dependent prostate cancer cells(LNCa P)and castration resistant prostate cancer cells(PC-3).Part two Study on the interaction between circular RNA cir-ITCH and mi R-17 in prostate cancer cellsObjective: To verify the interaction between cir-ITCH and mi R-17 in prostate cancer cells.Methods: LNCa P cells and PC-3 cells were treated with GFP transfection,cir-ITCH transfection,mi R-17 transfection,co-transfection of cir-ITCH and mi R-17,respectively.Quantitative reverse transcription polymerase chain reaction was used to detect the expression levels of cir-ITCH and mi R-17 in the transfected prostate cancer cells.Results: The expression levels of mi R-17 in LNCa P cells and PC-3 cells were significantly decreased after transfection of cir-ITCH,and cotransfection of cir-ITCH could significantly counteract the increased expression level caused by transfection of mi R-17.After transfection of mi R-17,the expression levels of cir-ITCH in LNCa P cells and PC-3 cells decreased significantly.Co-transfection of mi R-17 could significantly counteract the increased expression level caused by transfection of cir-ITCH.Summary: Cir-ITCH and mi R-17 restrained each other’s expression level in androgen dependent prostate cancer cells(LNCa P)and castration resistant prostate cancer cells(PC-3).Part three The effect and mechanism of cir-ITCH on the proliferation,migration and invasion of androgen dependent prostate cancer cells and castration resistant prostate cancer cellsObjective: To explore the effect and mechanism of cir-ITCH on the proliferation,migration and invasion of LNCa P and PC-3 cells.Methods: LNCa P cells and PC-3 cells were treated with GFP transfection,cir-ITCH transfection,mi R-17 transfection,co-transfection of cir-ITCH and mi R-17,respectively.Proliferation,migration,and invasion of PCa cells were evaluated using CCK-8,wound-healing assays and Transwell test.Western blot was used to detect the expression of androgen receptor,β-catenin,AKT,p-AKT,m TOR,and p-m TOR in prostate cancer cells treated with GFP transfection,cir-ITCH transfection,cir-ITCH transfection and exogenous androgen(dihydrotestosterone,DHT),cir-ITCH transfection and androgen receptor antagonists(Casodex).Results:1.Overexpression of cir-ITCH significantly inhibited the proliferation,migration and invasion of LNCa P and PC-3 cells.On the contrary,mi R-17 can promote the proliferation,migration and invasion of LNCa P and PC-3 cells.2.In LNCa P cells,overexpression of cir-ITCH down regulated the expression of AR,β-catenin,p-AKT and p-m TOR,which can be antagonized by DHT and enhanced by Casodex.In PC-3 cells,there was no AR expression.Overexpression of cir-ITCH down regulated the expression levels of β-catenin,p-AKT and p-m TOR.DHT and bicalutamide(Casodex)had no effect on this inhibition.Summary: Cir-ITCH can inhibit the proliferation,migration and invasion of both androgen dependent prostate cancer cells and castration resistant prostate cancer cells.Its tumor suppressing role may be achieved by inhibiting mi R-17 and regulating Wnt/β-catenin pathway and PI3K/AKT/m TOR pathway.This inhibition is independent of androgen receptor,so cir-ITCH has a potential value in the treatment of castration resistant prostate cancer.Conclusion The expression of cir-ITCH is significantly decreased in prostate cancer tissues and cells.Cir-ITCH and mi R-17 restrained each other’s expression level in androgen dependent prostate cancer cells(LNCa P)and castration resistant prostate cancer cells(PC-3).Cir-ITCH can inhibit the proliferation,migration and invasion of both androgen dependent prostate cancer cells and castration resistant prostate cancer cells.Its tumor suppressing role may be achieved by inhibiting mi R-17 and regulating Wnt/β-catenin pathway and PI3K/AKT/ m TOR pathway.This inhibition is independent of androgen receptor.