The Molecular Mechanism Of WNK2 Promotes Ovarian Cancer Proliferation And Migration By Targeting Ras/POU5F1B/AKT Pathway

Background: Ovarian cancer(OC)is one of the common gynecological malignancies,which is difficult to be diagnosed in the early stage and easy to cause chemotherapy resistance in the late stage.Therefore,OC is the first leading cause of death among gynecological malignancies.At present,the treatment of OC is tumor reduction surgery combined with chemotherapy based on paclitaxel/carboplatin,which is the current first-line treatment of OC.In recent years,the treatment of OC has been iterated and updated continuously.For example,Poly ADP ribose polyme Rase inhibitors(PARPs)have become the ideal choice for BRCA1/2 mutation carriers.However,despite various treatment options tried,the five-year survival rate for OC hovers between 30% and 40%.In order to improve the survival time of this disease,it is of great clinical significance to find new therapeutic targets.In 2000,Cobb et al.accidentally discovered a new class of serine/threonine kinase lacking lysine in subdomain II,namely,With no-lysine(K)kinase(WNK).At present,there are four WNK kinases(WNK1-WNK4)found in mammals,which play an important role in the occurrence and development of gastric cancer,colon cancer,and other tumors.Our team previously found that only WNK2 in the WNK family was highly expressed in OC through GEPIA database.Previous studies have shown that WNK2 is a tumor suppressor in liver cancer,glioma,gastric cancer,breast cancer,and colon cancer.Meanwhile,WNK2 is also a key effector molecule for many non-coding RNA regulating tumor progression.For example,in breast cancer,miR-370 promotes the proliferation of breast cancer cells by inhibiting WNK2.However,the functions and clinical significance of WNK2 in OC remain unknown.Therefore,this research intends to study the role and molecular mechanism of WNK2 in OC and whether miRNA regulates the proliferation of OC by regulating WNK2.Purposes: Provide a new molecular target for OC diagnosis and treatment.The details are as follows: 1.Reveal the roles of WNK2 by detecting the expression of WNK2 in OC cells,tissues,and validate the functional roles of WNK2 in vitro and in vivo.2.Explore the upstream and downstream target molecules of WNK2 in regulating OC progression.Methods: 1.Explored the differential expression of WNK2 in OC by GEPIA database.qRT-PCR,WB,and immunohistochemistry verified the expression,location and clinical significance of WNK2 in OC cells and tissues.2.Examined the functional roles of WNK2 by CCK8,colony formation and Transwell tests.3.Illustrated the molecular mechanisms regulated by WNK2 on OC progression by transcriptome sequencing,phosphorylation sequencing and verification experiments.4.Nude mice models were established to validate the functional roles of WNK2 and POU5F1 B in vivo.Results: 1.GEPIA database and IHC on microarray HOva C160Su01 verified the overexpression of WNK2 in OC tissues.No significant correlation exits between WNK2 levels and patients’ age or TNM stages(p>0.05),WNK2 expression is positively correlated with tumor malignancy(p=0.0068*).The Cox regression revealed the negative relationship between WNK2 and the patients’ survival time in serous ovarian adenocarcinoma(p=0.041*).2.qRT-PCR and WB verified the significant upregulation of WNK2 in three typical OC cell lines SKOV3,CAOV3,and A2780.Immunofluorescence revealed that WNK2 is significantly increased in cancer cell line SKOV3 contrary to ovarian epithelial cell line IOSE-80.And WNK2 exists in the cytoplasm of cancer cells.3.CCK8,colony formation and EDU proliferation assays illustrated the knockdown of WNK2 suppressed the malignant proliferation and migration of OC cells.Whereas,the overexpression of WNK2 promoted the malignant phenotypes of OC cells.4.Protein phosphorylation sequencing illustrated that when WNK2 was knocked down,the phosphorylation of most proteins was reduced and the most differentially phosphorylated proteins were enriched in the Ras pathway.Ras activation detection showed the knockdown of WNK2 suppressed Ras activation.5.RNA-seq sequencing predicts POU5F1 B as the candidate gene of WNK2,it was positively regulated by WNK2 both at mRNA and protein levels.Immunohistochemistry analysis presented conspicuous POU5F1 B upregulation in OC tissues than the adjacent normal tissues(p<0.05).Functional experiments(CCK-8,EdU,colony formation assays and Transwell analysis)proved POU5F1 B accelerates the proliferation and metastasis of OC cells.Moreover,POU5F1 B rescued proliferation and migration inhibition of WNK2 knockdown in vitro and in vivo successfully.6.qRT-PCR and WB proved WNK2 activates Ras/POU5F1B/AKT pathway.The knockdown of WNK2 suppressed the activation of p-AKT(S473).Moreover,overexpression of POU5F1 B reversed the inhibition of WNK2 knockdown on p-Akt(S473).Meanwhile,when WNK2 was overexpressed,the expression of POU5F1 B and activation of p-AKT(S473)was increased.After treating with salirasib,an inhibitor of Ras,the overexpression of POU5F1 B and activation of p-AKT(S473)disappeared.7.Targetscan,mirDIP and miRWalk databases predicts two binding sites between miR-324-3p and 3’UTR of WNK2.qRT-PCR verified the reduction of miR-324-3p in OC cells than the ovarian epithelial cell IOSE-80.In situ hybridization of HOva C070PT01 showed that miR-324-3p distinctly decreases in OC tissues.Luciferase assay indicated that miR-324-3p binds 3’UTR of WNK2 directly at 941-947 bp.8.When miR-324-3p increased in A2780 and CAOV3,the mRNA and protein level of WNK2 decreased.In contrast,the expression of WNK2 increased when miR-324-3p inhibition occurred.CCK-8,EdU,and colony formation assays showed miR-324-3p overexpression inhibits the proliferation of OC cells,while miR-324-3p inhibitor prompted the proliferation of OC cells.Rescue experiments confirms that WNK2 is the target of miR-324-3p.Conclusion: This research revealed WNK2 is overexpressed and exerts an oncogenic role in OC,which was also related to a poor prognosis in OC patients.As an oncogene,it promotes tumor cells proliferation and migration.We also found for the first time,that the Ras/POU5F1B/AKT pathway is the downstream effector of WNK2.And miR-324-3p,an OC suppressor,inhibits the tumor-promoting effects of WNK2.Our research revealed the new roles of WNK2 and found it may act as a new tumor marker and therapeutic target for OC patients in the future.