Genomic and epigenetic alterations deregulate microRNA expression in human epithelial ovarian cancer

Abstract

MicroRNAs (miRNAs) are an abundant class of small noncoding RNAs that function as negative gene regulators. miRNA deregulation is involved in the initiation and progression of human cancer; however, the underlying mechanism and its contributions to genome-wide transcriptional changes in cancer are still largely unknown. We studied miRNA deregulation in human epithelial ovarian cancer by integrative genomic approach, including miRNA microarray (n = 106), array-based comparative genomic hybridization (n = 109), cDNA microarray (n = 76), and tissue array (n = 504). miRNA expression is markedly down-regulated in malignant transformation and tumor progression. Genomic copy number loss and epigenetic silencing, respectively, may account for the down-regulation of approximately 15% and at least approximately 36% of miRNAs in advanced ovarian tumors and miRNA down-regulation contributes to a genome-wide transcriptional deregulation. Last, eight miRNAs located in the chromosome 14 miRNA cluster (Dlk1-Gtl2 domain) were identified as potential tumor suppressor genes. Therefore, our results suggest that miRNAs may offer new biomarkers and therapeutic targets in epithelial ovarian cancer.

Fig. 1.

Numerous miRNAs are down-regulated in late-stage or high-grade ovarian cancer. (A) Heat map showing the 44 miRNAs significantly down-regulated in late-stage relative to early-stage EOC. (B) Heat map showing the 13 miRNAs significantly down-regulated in high-grade relative to low-grade EOC. (C) Heat map showing the miRNAs significantly down-regulated in late-stage or high-grade serous EOC. (D) Venn diagrams of down-regulated miRNAs in different analyses.

Fig. 2.

DNA copy number deletions contribute to down-regulation of miRNAs. (A) DNA copy number status of 17 genomic loci containing miRNAs differentially expressed between IOSE cells and EOC cell lines. (Upper) Four up-regulated miRNAs. (Lower) Thirteen down-regulated miRNAs. Red lines indicate the designated cut-off for significant alterations (20%). (B) DNA copy number status of 16 genomic loci containing miRNAs significantly down-regulated in late-stage EOC. Red lines indicate the designated cut-off for significant alterations (20%).

Fig. 3.

Epigenetic alterations silence miRNA expression in ovarian cancer. (A) Heat map depicts expression of 24 miRNAs in IOSE cells and EOC cell lines analyzed by real-time RT-PCR. (B) Expression of the same 24 miRNAs in five EOC cell lines and HeLa cells after treatment with demethylating agent 5-aza-2′-deoxycytidine (5-Aza-CdR) and the histone deacetylase inhibitor 4-phenylbutyric acid (PBA) for 6 days.

Fig. 4.

miRNA deregulation affects mRNA transcripts. Histograms of negative natural logarithms of 4,096 P values derived from one-tailed Wilcoxon rank sum test applied to the distributions of each hexamer occurrence in the 3′ UTRs of all up-regulated versus unchanged mRNA transcripts.

Fig. 5.

Down-regulation of miRNA cluster at the Dlk1-Gtl2 domain is associated with poor survival. (A) Nonsupervised clustering of the eight Dlk1-Gtl2 domain miRNA expression signatures classifies 73 late-stage EOC in two distinct clusters (cluster 1, n = 38; cluster 2, n = 35). (B) Five-year survival of patients with advanced stage EOC whose tumors belong to cluster 1 (blue) or cluster 2 (green). (C) Summary of clinicopathologic characteristics of patients in the two clusters. (D) Examples of high and low proliferation index based on Ki67 immunohistochemistry staining in late-stage EOC. (E) Summary of proliferation index in tumors from the two clusters.