MicroRNA Gene Expression Signature Driven by <i>miR-9</i> Overexpression in Ovarian Clear Cell Carcinoma

<div><p>Previous studies have identified microRNA (miRNA) involvement in human cancers. This study aimed to elucidate potential clinical and biological associations of ovarian cancer-related miRNA gene expression profiles in high-grade serous carcinoma (HGSC) and ovarian clear cell carcinoma (OCCC). Accordingly, we investigated 27 patients with ovarian cancer (12 HGSC and 15 OCCC cases) using quantitative real-time reverse transcription polymerase chain reaction to determine the cancer-related miRNA expressions. Gene Cluster 3.0 was used for hierarchical clustering analysis, and differentially expressed miRNAs between HGSC and OCCC were identified by the class comparison analysis using BRB-ArrayTools. An unsupervised hierarchical clustering analysis identified two distinct miRNA expression clusters, with histological subtype-related significant differences in the associations between clusters and clinicopathological features. A comparison of miRNA expression in HGSCs and OCCCs identified five miRNAs (<i>miR-132</i>, <i>miR-9</i>, <i>miR-126</i>, <i>miR-34a</i>, and <i>miR-21</i>), with OCCCs demonstrating a statistically higher expression. Further investigation of the biological significance of <i>miR-9</i> overexpression in OCCC revealed that <i>miR-9</i> inhibition reduced the cell invasion ability and upregulated E-cadherin expression. Using a luciferase reporter assay, we further demonstrated the direct binding of <i>miR-9</i> to E-cadherin. Global cancer-related miRNA expression analysis identified statistically unique profiles that could discriminate ovarian cancer histotypes. In OCCC, <i>miR-9</i> overexpression may affect pathogenesis by targeting E-cadherin, thereby inducing an epithelial–mesenchymal transition. Therefore, <i>miR-9</i> may be a promising therapeutic target strategy for OCCC.</p></div

Clinicopathological Characteristics of 27 Patients with Ovarian Cancer.

<p>Clinicopathological Characteristics of 27 Patients with Ovarian Cancer.</p

Clinicopathological Characteristics of Patients with Ovarian Cancer.

<p>Clinicopathological Characteristics of Patients with Ovarian Cancer.</p

Validating original data by individual real-time RT-PCR analysis for both cohorts.

<p>Individual Taqman MicroRNA assays were used to analyze the relative expression of <i>miR-9</i> (A), <i>miR-132</i> (B), <i>miR-126</i> (C), and <i>miR-34a</i> (D) in the original 27 and additional 23 cases. Horizontal lines inside dots represent the median values.</p

Effects of <i>miR-9</i> knockdown on biological responses of OCCC cells.

<p>(A) JHOC-9 and OVISE cells transfected with either a miR-9 inhibitor or anti-miR-NC were subjected to an invasion assay. All experiments were repeated at least three times, and values are presented as means ± SDs. **P < 0.001. Representative images are shown. (B) E-cadherin, Vimentin, Fibronectin, and MMP-9 protein expressions in JHOC-9 and OVISE cells that were transfected with either miR-9 inhibitor or anti-miR-NC were analyzed by Western blotting. β-Actin expression was used as a loading control. (C) A luciferase reporter assay using the pMIR-REPORT^™ System was conducted to confirm direct binding of <i>miR-9</i> to <i>CDH1</i> (E-cadherin) 3′-UTR in JHOC-9 and OVISE cells. All experiments were repeated at least three times, and values are presented as means ± SDs. *P < 0.05, **P < 0.001.</p

Cancer-related miRNA expression profiles of 27 patients with ovarian cancer.

<p>An unsupervised hierarchical clustering analysis of 87 miRNA expression in 27 patients with ovarian cancer, including the calculated centered correlation distances and average linkages, identified two main clusters, A and B.</p

Differentially Expressed miRNAs in OCCC <i>vs</i>. HGSC.

<p>Differentially Expressed miRNAs in OCCC <i>vs</i>. HGSC.</p

Somatic Copy Number Alterations Associated with Japanese or Endometriosis in Ovarian Clear Cell Adenocarcinoma

<div><p>When compared with other epithelial ovarian cancers, the clinical characteristics of ovarian clear cell adenocarcinoma (CCC) include 1) a higher incidence among Japanese, 2) an association with endometriosis, 3) poor prognosis in advanced stages, and 4) a higher incidence of thrombosis as a complication. We used high resolution comparative genomic hybridization (CGH) to identify somatic copy number alterations (SCNAs) associated with each of these clinical characteristics of CCC. The Human Genome CGH 244A Oligo Microarray was used to examine 144 samples obtained from 120 Japanese, 15 Korean, and nine German patients with CCC. The entire 8q chromosome (minimum corrected p-value: <i>q</i> = 0.0001) and chromosome 20q13.2 including the <i>ZNF217</i> locus (<i>q</i> = 0.0078) were amplified significantly more in Japanese than in Korean or German samples. This copy number amplification of the <i>ZNF217</i> gene was confirmed by quantitative real-time polymerase chain reaction (Q-PCR). <i>ZNF217</i> RNA levels were also higher in Japanese tumor samples than in non-Japanese samples (<i>P</i> = 0.027). Moreover, endometriosis was associated with amplification of <i>EGFR</i> gene (<i>q</i> = 0.047), which was again confirmed by Q-PCR and correlated with <i>EGFR</i> RNA expression. However, no SCNAs were significantly associated with prognosis or thrombosis. These results indicated that there may be an association between CCC and <i>ZNF217</i> amplification among Japanese patients as well as between endometriosis and <i>EGFR</i> gene amplifications.</p></div

Comparison between Japanese and Koreans or Germans with regard to SCNAs on chromosome 20q13.2.

<p>Comparison between Japanese and Koreans or Germans with regard to SCNAs on chromosome 20q13.2.</p

Frequency of SCNAs gain or loss for 22 autosomes and the X chromosome.

<p>Frequencies (%) of copy number gain (right side of central axis, red) and copy number loss (left side of central axis, green) across the human genome are shown.</p