Study For Expression And Role Of ARRDC3/ARRDC3-ITBβ4 Pathway In Human Prostate Cancer

Background and purpose:Prostate cancer(PCa)is one of the common urinary tract malignant tumor,at present the morbidity and mortality of China showed a trend of rising year by year.Prostate cancer radical is the preferred treatment for early stage,endocrine therapy can be as the main treatment of late tumor.In recent years,Abitater as the representative of endocrine therapy and the molecular targeted therapy showed a certain advantage,but the effect has not yet been determined.However,almost all of the patients eventually develop into hormone dependence of prostate cancer,currently there is no effective treatments.Previous studies and our previous experiments have demonstrated that miR-30d is upregulated in prostate cancer and plays a key role in cancer progression.Therefore,we used gene expression profiling technique to analyze the gene expression profiles of LNCaP and DU 145 cell lines overexpressing miR-30d,and obtained a number of differentially down-regulated genes of miR-30d regulation,in which Arrestin domain-containing protein 3(ARRDC3)gene expression levels were significantly reduced.Contains Arrestin domain structure of protein 3(ARRDC3)is a member of the mammalian alpha inhibit protein family,has been identified as tumor suppressor genes,but its function in human prostate cancer has not been reported.Draheim KM and other studies have shown that ARRDC3 binds directly to the phosphorylated form of ITGβ4,leading to its internalization,ubiquitination and ultimately degradation,inhibiting breast cancer progression.The aim of this study was to determine the expression of ARRDC3 in prostate cancer and the correlation between ARRDC3 and the progression and prognosis of prostate cancer and to explore the role of ARRDC3-ITGβ4 pathway in prostate cancer.Methods:1.We constructed miR-30d down-expression and overexpression of LNCaP cell lines,Expression correlation between miR-30d and ARRDC3 was verified by qRT-PCR and Western blotting respectively.2.ARRDC3 expression was analyzed by immunohistochemistry in clinical prostate cancer tissue microarray.The relationship between the expression level of ARRDC3 and the clinical features and prognosis of prostate cancer patients was analyzed by statistical method.The relationship between the expression of ARRDC3 and the overall survival rate,biochemical recurrence survival rate was analyzed by survival curve analysis and Cox regression analysis,and to determine whether it can be used as a predictor of prognosis.3.We constructed DU 145 and LNCaP cell lines of ARRDC3 down-expression and overexpression,effect of ARRDC3 on the function of prostate cancer cells was analyzed by cell function experiment.4.To observe the effect of ARRDC3 on tumorigenesis of prostate cancer in vivo,we construct subcutaneous xenograft model by inoculation of ARRDC3 overexpressing DU 145 cells lines into subcutaneous tissue of nude mice.5.The expression of Ki67,MMP-9 and ITGβ4 in subcutaneous tumor tissues of nude mice were analyzed by immunohistochemistry.The expression of ITGβ4 in subcutaneous tumor cells of nude mice was detected by Western blotting.Results:1.We obtained a number of differentially down-expression genes in the gene expression profiling of miR-30d overexpression,and ARRDC3 was significantly downgraded in all down-expression genes.The results of qRT-PCR and Western blot analysis showed negative correlation between ARRDC3 and miR-30d.2.Tissue chip immunostaining analysis showed that low expression of ARRDC3 in prostate cancer tissue samples was significantly associated with high Gleason score(P = 0.045).Taylor database analysis ARRDC3 results showed that ARRDC3 down-regulated expression was associated with Gleason score,postoperative metastasis and biochemical recurrence(P = 0.0001,0.01,and 0.02,respectively).3.Kaplan-Meier and Log rank analysis showed that low expression of ARRDC3 was associated with poor prognosis in patients with prostate cancer(P = 0.010).COX regression model single factor and multivariate analysis showed that ARRDC3 is an independent predictor of biochemical recurrence in patients with prostate cancer.4.The expression of ARRDC3 could enhance the proliferation,migration and invasion of DU 145 and LNCaP cell lines(P<0.05).The expression of ARRDC3 could enhance the proliferation,migration and invasion ability of ARRDC3 cells(P<0.05).5.Animal experiments showed that overexpression of ARRDC3 could significantly inhibit the growth rate of subcutaneous xenografts in nude mice(P<0.05).Compared with the control group,the immunohistochemical scores of Ki67 and MMP-9 were significantly decreased(P<0.05),and it was confirmed that overexpression of ARRDC3 inhibited the proliferation and invasion of tumor cells.6.Western blot and immunohistochemical results showed a negative correlation between ARRDC3 and ITGβ4(P<0.05).Conclusion:1.ARRDC3 plays an anti-cancer effect in prostate cancer and inhibits the progression of prostate cancer and predicts the risk of postoperative biochemical recurrence and metastasis as an independent predictor.2.ARRDC3 can inhibit the progression of prostate cancer through ARRDC3-ITGβ4 pathway,and ARRDC3-ITGβ4 pathway may be used as a new therapeutic target for prostate cancer.