Additional file 1: of Risk prediction model for epithelial ovarian cancer using molecular markers and clinical characteristics

Table S1. Prognostic molecular markers in published studies. Table S2. The relationship between molecular markers and clinicopathologic characteristics. Figure S1. Single immunostaining of 15 molecular markers in epithelial ovarian cancer. Figure S2. Kaplan-Meier overall survival analyses of 15 molecular markers inepithelial ovarian cancer. (ZIP 108 kb

Citrate Synthase Expression Affects Tumor Phenotype and Drug Resistance in Human Ovarian Carcinoma

<div><p>Citrate synthase (CS), one of the key enzymes in the tricarboxylic acid (TCA) cycle, catalyzes the reaction between oxaloacetic acid and acetyl coenzyme A to generate citrate. Increased CS has been observed in pancreatic cancer. In this study, we found higher CS expression in malignant ovarian tumors and ovarian cancer cell lines compared to benign ovarian tumors and normal human ovarian surface epithelium, respectively. <i>CS</i> knockdown by RNAi could result in the reduction of cell proliferation, and inhibition of invasion and migration of ovarian cancer cells in vitro. The drug resistance was also inhibited possibly through an excision repair cross complementing 1 (ERCC1)-dependent mechanism. Finally, upon <i>CS</i> knockdown we observed significant increase expression of multiple genes, including <i>ISG15</i>, <i>IRF7</i>, <i>CASP7</i>, and <i>DDX58</i> in SKOV3 and A2780 cells by microarray analysis and real-time PCR. Taken together, these results suggested that CS might represent a potential therapeutic target for ovarian carcinoma.</p></div

<i>CS</i> silencing inhibits SKOV3 and A2780 cell invasion and migration in <i>vitro</i>.

<p>48 h after transfection, cell invasiveness was evaluated using the transwell assay and the Boyden Chamber test was used to measure the extent of cell migration. The number of invaded and migrated cells was counted using a bright-field microscope (200×). Representative images and the relative cell numbers were shown in (<b>A</b>) and (<b>B</b>). (<b>C</b>) p-FAK, MMP2 and Vimentin in <i>CS</i> knockdown cancer cells were analyzed using western blot. Mean ± SEM. *<i>P</i><0.05.</p

Gene expression profile related to drug resistance and apoptosis in SKOV3 and A2780 cells after <i>CS</i> silencing.

<p>(<b>A, B</b>) Ovarian cancer cells were treated with siRNA for 48 h, total RNA was extracted for gene expression analysis. Expression of <i>CASP7 (CASPASE7), IRF7 (</i>interferon regulatory factor 7), <i>DDX58 (</i>DEAD (Asp-Glu-Ala-Asp) box polypetide 58<i>)</i>, and <i>ISG15 (IFN</i>-stimulated gene 15<i>)</i> was increased significantly whereas <i>ATG12 (</i>autophagy related 12<i>)</i> expression was decreased in SKOV3 and A2780 cells after <i>CS</i> knockdown. <i>β-actin</i> was used as an internal control gene. Mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001. (<b>C</b>) The diagram was shown to identify potential signaling pathways modulated by CS.</p

<i>CS</i> silencing increases drug sensitivity in ovarian cancer cells.

<p>(<b>A</b>) Protein level of CS was examined in <i>CS</i>-silenced SKOV3 and A2780 cells. (<b>B</b>) SKOV3 and A2780 cells were treated with <i>CS</i> siRNA or negative siRNA for 48 h, then cells were treated with different concentrations of DDP for another 24 h. Cell viability was measured after incubation with CCK8 for 1.5 h. (<b>C</b>) 48 h after <i>CS</i> siRNA transfection, SKOV3 and A2780 cells were treated with 1 µg/ml DDP for 12 h and 1 h, respectively. Then cells were plated in 6-well plates, colonies were stained and counted after incubation for 8 days. Results shown were representative of three independent experiments. (<b>D</b>) SKOV3 and A2780 cells were treated with <i>CS</i> siRNA for 48 h, ERCC1 and γ-H2AX protein levels were compared between NC and siCS1078 group with β-tubulin used as a loading control. Mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001.</p

Up regulated expression of citrate synthase (CS) in human ovarian tumors and human ovarian tumor cell lines.

<p>(<b>A</b>) <i>CS</i> mRNA and (<b>B</b>) protein expression was assessed in normal human ovarian surface epithelium (HOSE), ovarian cancer cell lines, benign (n = 11) and malignant ovarian tumors (n = 21) using real-time PCR and western blot, respectively (B =  ovarian benign tumor, M =  ovarian malignant tumor). Mean ± SEM. **<i>P</i><0.01 and ***<i>P</i><0.001.</p

Enhanced Chemotherapeutic Efficacy of Paclitaxel Nanoparticles Co-delivered with MicroRNA‑7 by Inhibiting Paclitaxel-Induced EGFR/ERK pathway Activation for Ovarian Cancer Therapy

Chemotherapy-induced activation of cell survival pathways leads to drug resistance. MicroRNAs (miRNAs) post-transcriptionally regulate gene expression in many biological pathways. Paclitaxel (PTX) is one of the first-line chemotherapy drugs for ovarian cancer, and it induces the activation of the epidermal growth factor receptor (EGFR)/extracellular signal-regulated kinase (ERK) pathway that leads to tumor cell proliferation, survival, invasion, and drug resistance. MicroRNA-7 (miR-7) has the ability to suppress the EGFR/ERK pathway. To sensitize chemotherapy, we developed monomethoxy­(poly­(ethylene glycol))–poly­(d,l-lactide-<i>co</i>-glycolide)–poly­(l-lysine) nanoparticles for the simultaneous co-delivery of PTX and miR-7. The resulting PTX/miR-7 nanoparticles (P/MNPs) protect miRNA from degradation, possess a sequential and controlled release of drugs, improve the transfection efficiency of miRNA, decrease the half-maximal inhibitory concentration of PTX, and increase the apoptosis of ovarian cancer cells. The chemotherapeutic efficacy of PTX is prominently enhanced in vitro and in vivo via the inhibition of PTX-induced EGFR/ERK pathway activation by miR-7. Our studies in P/MNPs reveal a novel paradigm for a dual-drug-delivery system of chemotherapeutics and gene therapy in treating cancers

<i>CS</i> silencing affects AMPK/P38 MAPK pathway in ovarian cancer cell lines.

<p>(<b>A</b>) mRNA and <b>(B)</b> protein expression level of CS by real-time PCR and western blot in SKOV3 and A2780 cells after <i>CS</i> siRNA (100 nM) for 24 h and 48 h after transfection, respectively. (<b>C</b>) Decreased CS activity after 48 h transfection in SKOV3 and A2780 cells. (<b>D</b>) ATP level was examined 48 h after <i>CS</i> silencing. (<b>E, F</b>) p-AMPKα and p-p38 were analyzed in <i>CS</i>-silenced cancer cells by western blot. Mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001.</p

Regulatory network analysis of differentially methylated genes.

<p>A. regulatory network of differentially methylated genes between PCOS-NIR and PCOS-IR patients; B. regulatory network of differentially methylated genes between PCOS patients and healthy controls. The yellow nodes represent methylated genes and pink nodes represent normal genes.</p

<i>CS</i> silencing results in proliferation reduction in SKOV3 and A2780 cells.

<p>(<b>A</b>) Cell proliferation was measured in <i>CS</i>-silenced cancer cells and control groups. (<b>B</b>) p-ERK and <b>(C)</b> Bcl-2 expression were measured after <i>CS</i> siRNA for 48 h in SKOV3 and A2780 cells by western blot. β-tubulin was used as a loading control. (<b>D</b>) 48 h after <i>CS</i> siRNA, cells were treated with indicated concentrations of DDP (15 µg/mL or 10 µg/mL) for 24 h. Cleaved caspase 3 was measured by western blot. Mean ± SEM. *<i>P</i><0.05 and **<i>P</i><0.01.</p