ZMYND10,an Epigenetically Regulated Tumor Suppressor,exerts Tumor-suppressive Functions Via MiR145-5p/NEDD9 Axis In Breast Cancer

Objectives:Breast cancer is the most common malignant tumor in women in the world,and its morbidity ranks first in female malignant tumors.Breast cancer is divided into several major tumor subtypes according to estrogen or progesterone receptor expression and ERBB2 gene amplifycation.The diverse subtypes have different risk profiles and treatment strategies.The best treatment for each patient depends on the tumor subtype,anatomic cancer stage and patient's preferences.However,there are still great challenges in the clinical treatment of breast cancer due to its high heterogeneity and easy metastasis.At present,the silencing of tumor suppressor genes by DNA methylation has been proved to play an important role in the development of breast tumors.Epigenetic silencing of tumor suppressor gene ZMYND10 has been found to be closely related to the occurrence and development of lung cancer,nasopharyngeal cancer,gastric cancer and other cancers,but the biological function and potential molecular mechanism of ZMYND10 in breast cancer remain unclear.Thispaper aims to explore the biological function and potential molecular mechanism of ZMYND10 in breast cancer,so as to provide a new target for the clinical treatment of breast cancer.Methods:The mRNA expression level of ZMYND10 in paired breast tumor and adjacent non-tumor tissues were determined by qPCR.The protein expression and cellular localization of ZMYND10 were detected by Immunohistochemical staining.The expression level and methylation status of ZMYND10 in breast cancer cell lines,breast cancer tissues and normal breast tissues treated with demethylation reagent Aza with or without histone deacetylase TSA were determined by qPCR,qMSP and BGS.CCK8 assay,Transwell assay and flow cytometry analysis were used to detect the effect of down-regulated expression of ZMYND10 on proliferation,migration and invasion,apoptosis and cycle arrest of breast cancer cell lines.The ability of ZMYND10 to inhibit the proliferation of breast cancer cell lines in vivo was evaluated by xenograft in nude mice.qPCR,immunofluorescence,luciferase reporter assay and western blot were used to detect the effect of ZMYND10 on the regulation of NEDD9 expression.TargetScan software was used to predict the binding sites of miR145-5p and NEDD9.The regulation effect of ZMYND10 on miR145-5p expression was detected by qPCR.Luciferase reporter assay was used to detect the regulation effect of miR145-5p on NEDD9.Western blot,qPCR and Transwell assay were used to detect the negative effect ofmiR145-5p inhibitor on the signal axis of ZMYND10/ miR145-5p/NEDD9.Results:ZMYND10 was dramatically reduced in multiple breast cancer cell lines and tissues,which was associated with promoter hypermethylation.The expression of ZMYND10 was restored and hypermethylated levels were reduced in the promoter region after the treatment with the demethylated drug Aza+TSA.Overexpression of ZMYND10 in breast cancer cell lines with low expression of ZMYND10 can significantly inhibit the prolife-ration ability,induce cell apoptosis,block cell cycle in G2-M phase and greatly suppress the migration and invasion ability of breast cancer cell lines,suggesting that ZMYND10 plays an anti-cancer role in breast cancer.Furthermore,molecular mechanism studies indicated that ZMYND10 enhances expression of miR145-5p,which suppresses expression of NEDD9 protein through directly targeting the 3'-untranslated region of NEDD9 mRNA,thereby inhibiting breast cancer cell migration.Conclusions:Results from this study shows that ZMYND10 suppresses breast cancer tumorigenicity by inhibiting the miR145-5p/NEDD9 signaling pathway.This novel discovered signaling pathway may be a valid target for small molecules might help to develop new therapies to better suppress the breast cancer migration and invasion.