EZH2Participates In Malignant Biological Behavior Of Epithelial Ovarian Cancer Through Regulating The Expression Of BRCA1and The Identification Of Suitable Reference Genes For Normalization Of Quantitative Real-time PCR In Human FFPE Epithelial Ovarian Ti

ObjectiveThe purpose of this study is to investigate whether breast cancer1(BRCA1) is regulated by enhancer of zeste homo log2(EZH2) and plays a role in EZH2-mediated biological effects in epithelial ovarian cancer.MethodsTwo EZH2short hairpin RNAs (shRNAs) plasmid vectors were constructed, stable transfected into A2780cells and transient transfected into ES2and SKOV3. Quantitative real-time PCR, western blotting and immunocytochemistry were used to examine the mRNA and protein level of BRCA1after inhibition of EZH2. After the treatment of Akt-1activator Insulin-like Growth Factor-1(IGF-1), immunocytochemistry were further used to examine the translocation of BRCA1.Three different small interfere RNAs (siRNAs) for BRCA1were synthesized to transient knock down BRCA1in ovarian cancer cells with EZH2down-regulation or controls. Then MTT, flow cytometer and transwell assay were used to detect the function of BRCA1in EZH2mediated the cell proliferation, cell cycle and migration. MTT further evaluated the cisplatin cytotoxicity followed by EZH2or BRCA1inhibition in cisplatin resistant cell A2780/DDP simultaneously or separately.Results1. Depletion of EZH2increased BRCA1protein expression and promoted its nuclear translocation, but decreased BRCA1mRNA expression in epithelial ovarian cancer cells (A2780, SKOV3and ES2).2. Activation of Akt-1prevented BRCA1nuclear/cytoplasmic shuttling in A2780cells transfected with shEZH2s. 3. The proliferation and migration ability of A2780and ES2cells transfected with shEZH2s increased after combined inhibition of BRCA1.4. Down-regulation of EZH2or BRCA1sensitized A2780/DDP cells to cisplatin, whereas simultaneous inhibition of them only resulted in modest resensitization instead of showing any synergistic effect.ConclusionEZH2can modulate the expression of BRCA1and induce its cytoplasmic/nuclear translocation in epithelial ovarian cancer. BRCA1is required for the effects of EZH2down-regulation on biological behaviors of epithelial ovarian cancer cells. ObjectiveThe purpose of this study is to identify the suitable reference genes for normalization of quantitative real-time PCR in human FFPE epithelial ovarian tissue samples.MethodsWe performed a PubMed search using the MeSH terms "real-time PCR" and "ovarian cancer" and obtained128available articles published from January1st,2010to March10th,2013. Twelve frequently used housekeeping genes (ACTB, GAPDH,18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLPO, RPL13A, and B2M) were analyzed in50ovarian samples from normal, benign, borderline, and malignant tissues. Laser microdissection (LMD), an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA) and nonparametric (Kruskal-Wallis) tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes.ResultsEpithelial cells occupied a small percentage of the normal ovary. The expression of ACTB, PPIA, RPL13A, RPLPO, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLPO, and TBP) and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. ConclusionIn the study of epithelial ovarian tumors, we recommend the use of homogeneous tissues and multiple-reference normalization strategy for qPCR, e.g. the combination of ACTB, PPIA, RPLPO, and TBP. Whereas GAPDH, the most commonly used reference gene, is not recommended as a single reference gene.