NKAIN2 Inhibits Prostate Cancer Growth And Progression Through Suppressing Na,K-ATPase Expression And Activity

BackgroundProstate cancer is the most common malignant tumors in man from the western developed countries.The incidence of prostate cancer is rising year by year in China in recent years.Most prostate cancer,belong to relatively inert tumor,however,part of the prostate cancer progress quickly,and soon become threaten to patients lives.How to identify the quick progress patients,and the molecular mechanism of the proliferation and progression of prostate cancer,is of great significance to improve the survival rate of prostate cancer patients.NKAIN2 was first named T cell lymphoma fracture site related targets 1(T-cell lymphoma breakpoint associated target 1(TCBA1)),based on its interaction with potassium sodium pump beta subunit 1(Na,K-ATPase ?1)and was named the potassium sodium pump interaction factor 2,belong to the NKAIN transmembrane protein superfamily.The early study of our team using high-throughput SNP chip analysis found that NKAIN2 was often recurrently truncated and deleted in prostate cancer specimens,thus we speculated that NKAIN2 may be a tumor-suppressor genes in prostate cancer.Further by analyzing a large number of samples of prostate cancer,NKAIN2 often lost its function by truncation and deletion,especially in Chinese patients.And in vitro experiments show NKAIN2 can inhibit tumor cell proliferation,migration and invasion,and promote apoptosis in tumor cell lines.The role of Na,K-ATPase in cancer has caused wide public concern,many studies have found that its activity enhanced in a wide variety of cancer,and Na,K-ATPase inhibitors such as cardiac glycoside,has proved has inhibitory effect to many kinds of tumors including prostate cancer,and some inhibitors have entered clinical trials.Our preliminary study identified NKAIN2 play a role of tumor suppressor in prostate cancer,and as a Na,K-ATPase interaction factor,we speculated that it may play the role of tumor suppressor through inhibit Na,K-ATPase.MethodsBased on the above understanding,this study first used western blotting and Na,K-ATPase activity kit to detect the expression of Na,K-ATPase ?1 and ?1 subunit and Na,K-ATPase activities in prostate cancer cell lines,and by immunohistochemical detection to analyze the expression of Na,K-ATPase al and?1 subunit in prostate cancer tissue;Then Using immune coprecipitation to verify the interaction of NKAIN2 and Na,K-ATPase;Using the CRISPR/CAS9 knockout technology to build CAS9/SgRNA plasmid lentivirus vector and transfected 22RV1 cell line(NKAIN2 high expression),and by Western blotting and Cruiser enzyme knockout and mutation detection kit to confirm the knockout efficiency;Further through nude mice subcutaneous tumor generation experiment to verify the tumor suppressor role of NKAIN2 in vivo,at the same time by Western blotting deceted PI3K/Akt pathway activation after NKAIN2 knockout;Then,overexpressed and knockdown NKAIN2 in prostate cancer cells and used Western blotting and Na,K-ATPase activity kits to evaluate the expression of Na,K-ATPase ?1 subunit and Na,K-ATPase activities,thus analyzed the influence of NKAIN2 on Na,K-ATPase.Knockdown Na,K-ATPase ?1 subunit in prostate cancer cells and analyzed its role in prostate cancer cells growth and and progessionResults1.The expression of Na,K-ATPase ?1 and ?1 subunit and Na,K-ATPase activities in prostate cancer cells are much more higher than prostate epithelial cell;And by immunohistochemical detection of 30 pair prostate cancer tissue we found that the expression of Na,K-ATPase al and ?1 subuint is increased in prostate cancer tissues compared to adjacent normal tissue.2.Western blotting and Cruiser enzyme test results confirmed the 22RV1 NKAIN2 knockout cell lines,further nude mice subcutaneous tumor generation experiment showed that the tumor sizes generated by NKAIN2 knock out cell line are larger the negative control cells,and found that knockout of NKAIN2 could activate PI3K/Akt pathway.3.In PC-3 and 22 rv1 cells,the overexpression of NKAIN2 inhibited the expression of Na,K-ATPase ?1 subunit,on the contrary knockdown of NKAIN2 enhanced Na,K-ATPase ?1 subunit expression.4.In PC-3 and 22 rv1 cells,the overexpression of NKAIN2 inhibited Na,K-ATPase activity,and knockdown of NKAIN2 enhanced the activity of Na,K-ATPase.5.In PC-3 and 22 rvl cells,knockdown of Na,K-ATPase ?1 subunit decreased prostate cancer cell proliferation,migration,invasion and increased apoptosis.ConclusionThis study confirmed the inhibitory effect of NKAIN2 to the growth of prostate cancer in vivo.The expression of Na,K-ATPase ?1 subuint in prostate cancer cell lines and tissues,and Na,K-ATPase activity enhanced in prostate cancer lines.NKAIN2 may inhibit the proliferation and progression of prostate cancer through down-regulating the expression and activity of Na,K-ATPase.The findings may provide new theoretical basis for treatment of prostate cancer and evaluate,providing a new method for the clinical diagnosis and treatment of prostate cancer.