The Role Of TMEM16A Ca²⁺ Activated Chloride Channels In Proliferation And Migration Of Prostate Cancer Cells

Prostate cancer is a malignant tumor with a high incidence of males,the prevalence of the disease ranks second in men 's susceptibility to tumors worldwide,and its death rate ranks sixth.Early prostate cancer can be castrated for surgery,however,most patients will change to castration-resistant prostate cancer after treatment,which not only increases the difficulty of treatment but also increases the mortality rate.Therefore,to explore the generation and development mechanism of prostate cancer and to find new gene targets and drug inhibitors are essential for human health and the extension of patient life.Recent studies have found that a Ca²⁺-activated chloride ion channel TMEM16 A gene expression can participate in the regulation of certain biological functions of prostate cancer,but there is still controversy about its regulation and specific mechanism.TMEM16A?ANO1?is a member of the Anoctamin family.The gene is located in the CCND1-EMS1 region of human chromosome 11q13.This region is a tumor expansion region,and the occurrence of various cancers is closely related to the expansion of this region.Therefore,TMEM16 A can be used as one of the key objects in prostate cancer research.In this paper,prostate cancer cell lines PC-3,LNCap,DU145 and prostate epithelial cells RWPE-1 were selected,the expression of TMEM16 A in prostate cancer cells was analyzed by q PCR experiment,immunocytochemistry and Western Blot experiment;use immunofluorescence experiments to determine the localization of TMEM16 A expression;according to pharmacological inhibition and si RNA transfection,the role of TMEM16 A in the proliferation and migration of prostate cancer cells was analyzed by using CCK-8 experiment,scratch experiment and Transwell experiment,and observe whether androgen DHT can play a role in the growth and metastasis of prostate cancer cells by affecting the expression of TMEM16 A.The purpose of this paper is to explore the specific role and related mechanisms of TMEM16 A in the proliferation and migration of prostate cancer cells,to provide new ideas for the research on the signal pathways related to the promotion of prostate cancer cells' proliferation and migration,and lay the foundation for TMEM16 A as a new target for the treatment of castration-resistant prostate cancer.Experimental results:1.The results of immunofluorescence experiment shows that TMEM16 A is mainly located on the plasma membrane of prostate cancer cells,and a small part exists in the cytoplasm.2.Real-time fluorescence quantitative PCR is consistent with the results of WB.TMEM16 A is expressed in RWPE-1,PC-3,DU145 and LNCap.The order of TMEM16 A expression in these four cell lines is PC-3> DU145> LNCap> RWPE-1.3.The CCK-8 experiment was used to detect that when different concentrations of TMEM16 A selective inhibitors T16Ainh-A01 and Ca CCinh-A01 were added,the activity of TMEM16 A was reduced due to the weakened ion channel activity,thereby inhibiting the proliferation of prostate cancer cells.In PC-3,the inhibitors inhibites its proliferation in a dose-dependent and time-dependent manner;however,at 10 ?M,these two inhibitors only started to inhibit LNCap and DU145 cells.4.Through cell scratch experiment and Transwell experiment observation,when adding different concentrations of TMEM16 A inhibitors T16Ainh-A01 and Ca CCinh-A01,the migration rate of cells was inhibited.PC-3 was inhibited in a concentration and time-dependent manner.For DU145 cells,the inhibitory effect was significant when T16Ainh-A01 concentration was at 16 ?M;Ca CCinh-A01 concentration was at 8 ?M.5.After knocking down the expression of TMEM16 A with si RNA at a final concentration of 50 n M,it can inhibit the proliferation and migration of PC-3 cells.6.The male hormone DHT is used to participate in the expression of TMEM16 A in the androgen-dependent prostate cancer cell LNCap.Different concentrations of DHT are used to stimulate LNCap cells,and the expression level of mRNA and protein is observed to change.The results of q PCR and WB experiment show that DHT can induce TMEM16 A overexpression in LNCap cells,and the effect is significant when the concentration of DHT is at 1 ?M.7.The results of the CCK-8 experiment and the scratch experiment show that the addition of TMEM16 A inhibitors T16Ainh-A01 and Ca CCinh-A01 and the androgen receptor AR antagonist bicalutamide BCT can inhibit the proliferation and migration of prostate cancer cells by the reason of overexpression of TMEM16 A in LNCap which adding DHT.8.In order to study the specific mechanism of TMEM16 A expression in the proliferation and migration of prostate cancer cells,the proteins Cyclin D1,Cyclin E1,E-cadherin,Vimentin related to proliferation and migration mediated by the NF-?B pathway were selected.Observe the changes in the expression of these proteins after knocking down the expression of TMEM16 A,and detect the protein changes of NF-?B?p65?.The results showes that:?1?After knocking down the expression of TMEM16 A in PC-3 cells,the expressions of Cyclin D1,Cyclin E1,and Vimentin were down-regulated,while the expression of E-cadherin was up-regulated;?2?After the expression of TMEM16 A decreased,the expression of p65 also decreased.In summary,the results of the study shows that inhibiting the activity and expression of Ca²⁺-activated chloride channel TMEM16 A in prostate cancer cells can inhibit the proliferation and migration of prostate cancer cells.And the selective inhibitor of TMEM16 A can also inhibit the proliferation and migration of cells involved in DHT.In this study,it was found that by knocking down the expression of TMEM16 A in prostate cancer cells,the expression of genes related to proliferation and migration mediated by NF-?B also changed which is to reveal the role of TMEM16 A overexpression in promoting the development of prostate cancer and the mechanism provides important theoretical significance.