Profile of differentially expressed miRNAs in high-grade serous carcinoma and clear cell ovarian carcinoma, and the expression of miR-510 in ovarian carcinoma

Abstract

Improved insight into the molecular and genetic profile of different types of epithelial ovarian cancer (EOC) is required for understanding the carcinogenesis of EOC and may potentially be exploited by future targeted therapies. The aim of the present study was to identify a unique microRNA (miRNA) patterns and key miRNAs, which may assist in predicting progression and prognosis in high‑grade serous carcinoma (HGSC) and clear cell carcinoma (CCC). To identify unique miRNA patterns associated with HGSC and CCC, a miRNA microarray was performed using Chinese tumor bank specimens of patients with HGSC or CCC in a retrospective analysis. The expression levels of four deregulated miRNAs were further validated using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) in an external cohort of 42 cases of HGSC and 36 cases of CCC. Kaplan‑Meier analysis was performed to analyze the correlation between the expression levels of the four miRNAs and patient prognosis. Among these validated miRNAs, miR‑510 was further examined in another cohort of normal ovarian tissues, as well as the HGSC, low‑grade serous carcinoma (LGSC) and CCC specimens using RT‑qPCR and in situ hybridization. The results revealed that, of the 768 miRNAs analyzed in the microarray, 33 and 50 miRNAs were significantly upregulated and downregulated, respectively, with at least a 2‑fold difference in HGSC, compared with CCC. The quantitative analysis demonstrated that miR‑510 and miR‑129‑3p were significantly downregulated, and that miR‑483‑5p and miR‑miR‑449a were significantly upregulated in CCC, compared with HGSC (P<0.05), which was consistent with the microarray results. Kaplan‑Meier analysis revealed low expression levels of miR‑510 and low expression levels of miR‑129‑3p, advanced International Federation of Gynecology and Obstetrics (FIGO) stage, lymphatic metastasis and that HGSC was significantly associated with the poorer overall survival rates (P<0.05). The expression of miR‑510 was significantly higher in the LGSC and CCC tissues, compared with the HGSC and normal ovarian tissues. The results of the present study suggested that different subtypes of EOC have specific miRNA signatures, and that miR‑510 may be involved differently in HGSC and CCC. Thus, miR‑510 and miR‑129‑3p may be considered as potential novel candidate clinical biomarkers for predicting the outcome of EOC.

Figure 1

Unsupervised hierarchical clustering of miRNAs and samples from the microarray data. The miRNA clustering tree is on the left and the sample clustering tree is above. The color scale at the top indicates the relative expression level of an miRNA across all samples. Red indicates that the expression level is higher than the mean; blue indicates that the expression level is lower than the mean. Each row represents one miRNA and each column represents one sample. Clustering was performed using log10 (fold change) ratios, which passed the filtering criteria across all samples (standard deviation <1.0). The heat map contained 102 unique miRNAs, which were differentially expressed in patients with high-grade serous carcinoma and clear cell carcinoma.

Figure 2

Comparison between RT-qPCR results and microarray results. The transverse bars indicate the median value, error bars indicate the SD, microarray results are presented as log10²-averageDC and the data are expressed as the mean ± SD of HGSC (n=20) and CCC (n=6). The RT-qPCR data are presented as the logarithmic mean expression level relative to HGSC, and the data are expressed as the mean ± SD of HGSC (n=42) and CCC (n=36). Each sample was assessed in triplicate. Statistical significance was determined using a Mann-Whitney test. The microarray and RT-qPCR results revealed significant differences between HGSC and CCC. The expression levels of miR-510 and miR-129-3p were significantly downregulated, those of and miR-483-5p and miR-449a were significantly upregulated in HGSC, compared with CCC (P<0.05). RT-qPCR, reverse transcription-quantitative polymerase chain reaction; CCC, clear cell carcinoma; HGSC, high-grade serous carcinoma; SD, standard deviation.

Figure 3

Kaplan-Meier curves of the overall survival rates of patients with EOC, stratified by expression levels of miR-510 and miR-129-3p. Reverse transcription-quantitative polymerase chain reaction data of miR-510 and miR-129-3p were separated into high and low expression values. miR-129-3p was separated by the median value of the miRNA levels in the tumor samples. MiR-510 was separated by the expression level which can give the greatest significance in log-rank test in the Kaplan-Meier analysis. The reference was 2 for miR-510. The patients with EOC were divided into groups exhibiting high expression (solid line) and low expression (dotted line) levels of miR-510 and miR-129-3p. The survival duration of the patients with EOC was associated with miRNA expression levels. Lower expression levels of miR-510 (P=0.048) and miR-129-3p (P=0.039) were associated with poorer overall disease-specific survival rates. EOC, epithelial ovarian cancer; miR, microRNA; Cum, cumulative. High, high expression level group; low, low expression level group.

Figure 4

Expression levels of miR-510 in normal ovarian tissue, HGSC, LGSC and CCC samples. The transverse bars indicate the median value of the results of the reverse transcription-quantitative polymerase chain reaction of miR-510, error bars indicate 95% confidential intervala. The expression levels of miR-510 in the CCC and LGSC samples were significantly higher, compared with the normal ovarian tissue and HGSC samples. No significant difference was identified between the expression levels of miR-510 in the CCC and LGSC samples (P=0.198) or the HGSC and normal ovarian tissue samples (P=0.860). miR, microRNA; EOC, epithelial ovarian cancer; HGSC, high-grade serous carcinoma; CCC, clear cell carcinoma; LGSC, low-grade serous carcinoma; OSE, ovarian surface epithelium.

Figure 5

In situ hybridization of miR-510 in LGSC, CCC and HGSC. The signal was visualized by BCIP/NBT solution and the nuclei were counterstained with nuclear fast red. (A) Malignant cells in LGSC exhibited a clear blue signal in the cytoplasm and nucleus. (B) Malignant cells in CCC exhibited a clear blue signal. (C) HGSC exhibited a weak blue signal only. Magnification, ×200. HGSC, high-grade serous carcinoma; LGSC, low-grade serous carcinoma; CCC; clear cell carcinoma; miR, microRNA.