Functional And Mechanistic Research On The Roles Of MELK In Endometrial Carcinoma

Objectives: Endometrial carcinoma(EC)is one of the most common malignancies in the female reproductive system.The incidence of EC increases every year and it shows a younger trend.Although it has a favourable overall prognosis,the prognosis of patients with recurrent and metastatic EC are poor.Conventional treatments have affected the quality of life of the patients.Also,young patients have urgent requirements for the preservation of fertility.Therefore,molecular targeted therapy has become the key to solving the above problems.This study aims to explore the role and function of Maternal embryonic leucine Zipper Kinase(MELK)involving in the progression of EC.The study will help us to deepen the understanding of the pathogenesis of EC and find new prognostic biomarkers and their related inhibitors to explore new ideas on molecular targeted therapy for EC.Contents and Methods:(1)Analysis the mRNA expression of MELK in EC by using TCGA and GEO databases;Analysis the relationship between MELK expression and clinicopathological parameters in the database.(2)The protein level of MELK was detected by immunohistochemistry on EC tissue microarray,and the relationship between MELK expression and clinicopathological parameters was analyzed.(3)RNAi technology was used to knockdown the expression of MELK(siMELK);CCK-8 assay and clone formation assay was used to detect the effect of siMELK on cell proliferation of EC cell lines HEC1 A and AN3CA;Wound healing assay was to detect cell migration of EC cell lines after MELK was knockdown.Flow cytometric analysis was to examine cell cycle.Subcutaneous tumorigenesis assay was used to detect the effect of shMELK cells(HEC1A)in vivo.(4)Chromatin immunoprecipitation(ChIP)and luciferase reporter gene assay was used to confirm transcriptional factor-E2F1 regulating MELK expression by binding the MELK promotor region.(5)Co-IP and immunofluorescence assays verified that MELK interacted with MLST8;Western blotting assay confirmed MELK can activate mTORC1/2 signaling to promote tumorigenesis.(6)The effect of OTSSP167 as an inhibitor of MELK was used to detect cell proliferation of HEC1 A and AN3 CA cells.Results:(1)At mRNA and protein levels,MELK expression in EC tissues was higher than that in normal control tissues;MELK expression was higher in late stages,high grades and serous EC;High MELK expression group patients tend to have a shorter overall survival time and disease-free survival time.;high expression of MELK was one independent risk factors for EC.(2)MELK knockdown decreased the ability of cell proliferation and migration in vitro;Cell cycle was arrested in G2/M phase;Subcutaneous tumorigenesis assay confirmed that shMELK cells reduced EC cell growth in vivo.(3)E2F1 can bind to the MELK promoter region and promote the transcriptional expression of MELK.(4)MELK activated the mTORC1/2 signaling by interacting with MLST8,thereby promoting cell growth and proliferation.(5)OTSSP167 can suppress the proliferation of EC cells by inhibiting the expression of MELK.Conclusions: MELK can promote the progression of EC via the E2F1-MELK-mTORC signaling axis,which is a potential therapeutic target for EC;OTSSP167 can be a potential new targeted drug for EC treatment by targeting MELK.