Mechanism Of Lysine Specific Demethylase 1 In Autophagy Of Ovarian Cancer Cells

Objective:This thesis is to determine expression of lysine-specific demethylase 1(LSD1)and relationship between LSD1 and autophagosomal protein LC3 in human ovarian cancer tissues and explore the role of LSD1 in autophagy of human ovarian cancer cells,thus further understand the pathogenesis of ovarian cancer and develop novel molecular targeted therapy for ovarian cancer.Methods: 1?Expression of the LSD1 and LC3 genes in the cancer genome atlas(TCGA)and theOncomine databases was bioinformatically analyzed with Kaplan-Meier plotterand other analytic tools.2?The correlation between LSD1 and LC3 gene expression was determined by GEPIAsoftware.3?The GST-LC3 B pulldown was used to detect interaction of LC3 with the LSD1SWIRM domain.4?Subcellular localization of LSD1 and LC3 in ovarian cancer cells was detected byimmunofluorescence staining.5?The methylation level of LC3 protein in ovarian cancer HO8910 and SKOV3 cellsand the effect of LSD1 on the LC3 methylation were detected by proteinimmunoprecipitation method.6?The effect of LSD1 knockdown on the stability of LC3 protein was detected bywestern blotting under treatment of the protein synthesis inhibitor cycloheximide(CHX).7?Regulation of LC3 by LSD1 was detected using Western blot under treatment ofChloroquine(CQ)or MG132,western blotting.8?The effect of LC3 on the demethylase activity and expression of LSD1 was detectedusing Western blot.Results: 1?Bioinformatical analysis showed that expression of LSD1 was negatively correlatedwith expression of LC3 in ovarian cancer patients,and LSD1 was highly expressedin ovarian cancer tissues instead of surrounding normal tissues.Patients with highexpression of LSD1 had poor prognosis,while those with high expression of LC3had better prognosis.2?The LSD1 SWIRM domain is the LC3 binding site.3?LSD1 and LC3 were colocalized in the nucleus of ovarian cancer HO8910 cells.4?LC3 was methylated in ovarian cancer HO8910 and SKOV3 cells,and thedemethylase LSD1 may mediate the demethylation of LC3.5?LSD1 knockdown significantly increased the expression level of LC3 protein inovarian cancer HO8910 and SKOV3 cells,while LC3 may negatively regulate thedemethylase activity of LSD1.Conclusions: 1?There was a negative correlation between expression of the LSD1 and LC3 genesin ovarian cancer patients.LSD1 was highly expressed in ovarian cancer tissues.Patients with high LSD1 expression had poor prognosis,while patients with highLC3 expression had better prognosis.2?LSD1 interacts with the autophagosomal protein LC3 and mediates demethylationof LC3,thereby affecting the stability of LC3 protein and regulating the autophagyprocess in ovarian cancer cells.This study has revealed a novel signaling pathwayby which LSD1 regulates autophagy in ovarian cancer cells.