The Study On The Role Of Wnt5a In Vasculogenic Mimicry Formation And The Mechanism In Prostate Cancer

Objective:The incidence of prostate cancer in China is increasing year after year,and the proportion of progressive prostate cancer in China is higher than that in Europe and America.The treatment of progressive prostate cancer is difficult.The key to solve this problem is to explore its mechanism and therapeutic targets.Vasculagenic mimicry(VM)is a special blood supply pattern in solid tumors.It is a lumen like structure composed of tumor cells.Its appearance indicates the high malignancy and poor prognosis,and is closely related to tumor progression and metastasis.The formation of VM is closely correlated to epithelial mesenchymal transformation(EMT)and cancer stem cells(CSCs).At present,there is a lack of exploration on the progression mechanism of prostate cancer from this aspect.Therefore,it is of great importance to explore the formation mechanism of VM,and the treatment methods to block VM formation.Studies have shown that CSCs are the root of tumor occurrence,development,metastasis and drug resistance.Furthermore,EMT is a process of epithelial derived cells transforming into mesenchymal derived cells under specific physiological and pathological conditions.EMT can promote tumor cells to obtain and maintain stemness,and it is the key step to form VM.Wnt signaling is important in regulating CSCs and EMT.Wnt5a,a non-classical signaling pathway ligand of Wnt signaling,can promote the formation of VM in lung and ovarian cancer,but its regulatory effect on VM in prostate cancer has not been reported.Therefore,the aim of this study was to investigate the role of Wnt5a in regulating VM,EMT and CSCs in prostate cancer,and further explore its molecular signaling pathways.Methods:(1)Immunohistochemical staining(IHC)was used to analyze the expression of Wnt5a in50 prostate cancer samples and paired benign prostatic hyperplasia samples,and expression of VM,EMT and CSCs markers in prostate cancer samples were also detected by IHC.In addition,CD34/PAS double staining was used to judge the formation of VM.Analyze the relationship between Wnt5a and VM,as well as VM,EMT and CSCs markers.The clinicopathological data were further used to analyze the relationship between Wnt5a and the tumor size,invasion,metastasis and pathological grade of prostate cancer.(2)The expression of Wnt5a,VM,EMT and CSCs markers in prostate cancer cell lines PC-3,LNCa P,DU145 and prostate immortalized normal epithelial cell RWPE-1 were detected by q RT-PCR and Western blot(WB).(3)A prostate cancer cell line stably knockdown or overexpressing Wnt5a was constructed.The transfection effect was detected by rescue experiment,q RT-PCR and WB.(4)The relationship between Wnt5a and VM markers was analyzed by q RT-PCR and WB.The effect of Wnt5a on VM formation in prostate cancer cells was detected by Matrigel three-dimensional culture.(5)The relationship between Wnt5a and the expression of markers of EMT was analyzed by q RT-PCR and WB.The effect of Wnt5a on EMT was further evaluated by observing cell morphology and intercellular junction,scratch test and Transwell test.(6)The relationship between Wnt5a and the CSCs markers was also analyzed by q RT-PCR and WB,and the role of Wnt5a on the characteristics of prostate cancer CSCs was further evaluated by CCK-8 experiment,plate clone formation experiment and flow cytometry.(7)The downstream pathway involved in Wnt5a regulation of VM was screened by using a variety of Wnt signal pathway inhibitors,and explore the signal cascade between Wnt5a and its downstream pathway.(8)The role and mechanism of Wnt5a in regulating VM of prostate cancer was further verified by constructing orthotopic prostate cancer transplanted tumor model in nude mice.Results:(1)The expression of Wnt5a in prostate cancer was significantly higher than that in benign prostatic hyperplasia(P=0.016),and its expression was positively correlated with t PSA(P=0.010),tumor proportion(P=0.002),Gleason score(P<0.001),vas deferens invasion(P=0.044)and lymphatic metastasis(P=0.044).(2)The expression of Wnt5a in prostate cancer was positively correlated with the existence of VM(P=0.008),and was positively correlated with the expression of VM marker VE-cadherin(P=0.013).(3)The expression of Wnt5a in prostate cancer was positively correlated with Vimentin(P=0.005)and CSCs marker(CD133)(P=0.012).(4)VM was also positively correlated with Vimentin(P=0.032),and CD133(P=0.008),and VM was positively correlated with tumor proportion(P=0.041),Gleason score(P=0.001),vas deferens invasion(P=0.014)and lymphatic metastasis(P=0.014).(5)DU145 cells and LNCa P cells were selected for further study according to their characteristics.Then DU145 cell lines with knockdown of Wnt5a and LNCa P cell lines with high Wnt5a expression were successfully constructed.(6)Wnt5a can up regulate the expression of VM markers(VE cadherin and MMP-9)and promote the formation of VM.(7)Wnt5a can up regulate the expression of Vimentin,while down regulate the expression of E-cadherin,and make the connection between prostate cancer cells from tight to loose.At the same time,Wnt5a can enhance the migration and invasion of prostate cancer cells.(8)Wnt5a can up regulate the expression of CSCs markers(CD133,Nanog,Sox2 and Oct4),enhance the proliferation and clonogenic ability of prostate cancer cells,and increase CD133~+cells proportion.(9)Wnt5a can activate Jnk signaling pathway by phosphorylation,so as to improve VM formation ability and stem cell characteristics of prostate cancer cells,and promote EMT process.(10)Wnt5a/Pi3k/Cdc42/Jnk/c-Jun signal cascade is the molecular mechanism by which Wnt5a regulates VM,EMT and CSCs of prostate cancer cells.(11)Animal experiments further verified that Wnt5a/Jnk signaling pathway plays a regulatory role in VM,EMT and CSCs of prostate cancer.Conclusion:(1)The high expression level of Wnt5a in human prostate cancer is related to the invasion,metastasis and malignancy of prostate cancer.(2)Wnt5a can promote the EMT of prostate cancer cells,and promote its invasion and metastasis.(3)Wnt5a can regulate stemness of prostate cancer cells and enhance its tumorigenicity and clonogenic ability.(4)Wnt5a can promote the formation of VM by promoting the EMT and CSCs characteristics of prostate cancer cells,and play an important role in the progression of prostate cancer.(5)Jnk pathway is the downstream signaling of Wnt5a,and Wnt5a can phosphorylate Jnk/c-Jun signaling pathway by activating Pi3k and Cdc42,and then regulate downstream target factors.