Depletion Of Dicer Promotes Epithelial Ovarian Cancer Progression By Elevating PDIA3 Expression

ObjectivesDicer is an essential component of the miRNA processing machinery whose low expression is associated with advanced stage and poor clinical outcome in epithelial ovarian cancer (EOC). The aim of this study was to investigate the functional relevance of Dicer in epithelial ovarian cancer and to identify its downstream effectors.Methods1. Dicer expression was depleted in three EOC cell lines (A2780, CAOV3 and SKOV3) by shRNA transfection. Gene expression was assessed using quantitative PCR and western blotting.2. Cell proliferation and migration were evaluated using EdU assay, colony formation assay, wound-healing assay and transwell assay.3. Two-dimensional gel electrophoresis combined with mass spectrometry was used for proteomic profiling. The functions of the identified proteins and their interactions were bioinformatically analyzed.4. Protein disulfide-isomerase A3 (PDIA3) was down-regulated by siRNA. MicroRNAs expression was assessed using quantitative PCR.5. Nude mice models were used to verify the interaction between Dicer and PDIA3 and to investigate their roles in EOC progression.6. Epithelial-mesenchymal transition (EMT) markers (vimentin and E-cadherin) and proliferation activity marker (Ki-67) of ovarian cancer xenografts were measured by immunohistochemistry.Results1. Dicer depletion promoted ovarian cancer cell proliferation and migration accompanied by a global up-regulation of proteins.2. Twenty-six proteins, seven up-regulated and nineteen down-regulated, were identified via two-dimensional gel electrophoresis combined with a liquid chromatography/tandem mass spectrometry method (LC-MS/MS).3. Based on proteomics screening and bioinformatics analysis, PDIA3 was selected as a potential downsteam target protein of Dicer for further study. Moreover, the miR-148a and miR-494 targeting PDIA3 were decreased in cells with Dicer depletion.4. PDIA3 repression could significantly relieve the proliferation-and migration-promoting effect mediated by Dicer depletion in vitro and in vivo.5. Immunohistochemistry showed an increase in the mesenchymal marker vimentin companied by a decrease in the epithelial marker E-cadherin and increased Ki-67 staining in the shDicer xenografts compared with the shNC group. But these effects were reversed in shDicer-transfected ovarian cancer cells co-transfected with siPDIA3.ConclusionsLow expression of Dicer contributes to epithelial ovarian cancer progression by elevating PDIA3 expression.