Clinical Impact of microRNAs Associated With Cancer Stem Cells as a Prognostic Factor in Ovarian Carcinoma

Abstract

Background: Ovarian carcinoma is a highly lethal gynecological malignancy due to its frequent relapses and adoption of chemoresistance. To develop new biomarkers for disease progression in ovarian carcinoma, CSCs, which are considered to contribute to disease relapse and metastasis, were isolated from human ovarian carcinoma tissues, and differentially expressed microRNAs (miRNAs) in CSCs were identified and assessed the clinical implication of expression of these miRNAs. Methods: Primary cancer cells derived from human ovarian carcinomas were cultured and spheroid-forming cells (SFCs) were isolated. Profiles of miRNA expression in CSC-like SFCs were identified by miRNA microarray and the results were validated by quantitative real-time RT-PCR (qRT-PCR). We also assessed the correlations between miRNA expression levels and clinicopathological parameters in ovarian carcinomas. Results: Five miRNAs (miR-5703, miR-630, miR-1246, miR-424-5p, and miR-320b) were significantly dysregulated in CSC-like SFCs compared with primary cancer cells. The qRT-PCR showed that miR-5703 and miR-1246 expression was significantly higher in ovarian cancer cells than in normal control cells, whereas the miR-424-5p level was significantly lower. Decreased expression of miR-424-5p was significantly associated with distant metastasis in high stage (stage IIII & IV) carcinomas (35.5% vs. 72.2%, respectively, p=0.013) Conclusion: Taken together, miR-5703, miR-630, miR-1246, miR-424-5p, and miR-320b are useful markers for enriching ovarian CSCs. Decreased expression of miR-424-5p in ovarian carcinoma might be a putative biomarker for distant metastasis in ovarian carcinoma.

Keywords: Cancer stem cell; Ovarian carcinoma; miR-424-5p, Prognosis.; microRNA.

Figure 1

MicroRNA microarray analysis of spheroid-forming cells (SFCs) and primary cancer cells from cultured ovarian serous carcinoma (OSC) cells. (A) Hierarchical clustering of 37 significantly altered miRNAs with a ≥2.0-fold difference in expression between OSC SFCs and primary cancer cells. The clustered expression data are displayed on a heat map, with individual tumors and miRNAs listed on x- and y-axes, respectively. The x-axis indicates four SFC samples and their respective primary cancer cell samples. (B) Twenty-four miRNAs were significantly upregulated, whereas 13 miRNAs were downregulated, in the four SFC samples when compared with the control. Individual blocks are color-coded to indicate changes in miRNA expression: red indicates upregulation, green indicates downregulation, and black indicates no change. SCN, cultured primary cancer cell; SFC, spheroid-forming cells. (C) KEGG pathway analysis of genes targeted by the dysregulated miRNAs in CSC-like SFCs compared with primary OSC cells. Genes targeted by five or more miRNAs were selected for pathway enrichment analysis. The enrichment score of each pathway is expressed as -log (P-value).

Figure 2

Comparison of miRNA expression levels (miR-5703, miR-630, miR-1246, miR-424-5p, and miR-320b) in OSCs using two approaches; namely, miRNA microarray analysis and qRT-PCR. (A) The left panel shows the relative miRNA expression levels determined by miRNA microarray analysis. Five miRNAs were dysregulated in CSC-like SFCs when compared with primary cancer cells. The right panel shows relative miRNA expression levels determined by qRT-PCR. The expression profiles of miR-5703 (upregulated), miR-424-5p, and miR320b (both downregulated) were similar between OSC and SFC samples. (B) The expression levels of candidate miRNAs in OSCs relative to fallopian tubes (control). miR-5703 and miR-1246 expression levels were significantly higher in OSCs than in fallopian tubes, whereas the miR-424-5p expression level was lower in OSCs than in the control. Data are presented as the mean ± SD. (*P < 0.05).