Long non-coding RNAs (lncRNAs)
More than 80% of risk SNPs occur in non-protein coding regions. Post-GWAS follow-up studies are beginning to identify the underlying mechanisms, but it is firmly established that some of this is due to inter-individual differences in non-coding RNAs (ncRNAs). Non-coding RNAs (ncRNAs) resemble protein-coding transcripts but without functional open reading frames and are typically classified according to size; thus small ncRNAs are less than 200 nucleotides in length whereas lncRNAs contain at least 200 nucleotides. lncRNAs are the most common type of ncRNA and influence cancer initiation and progression in several ways, including chromatin remodeling, transcriptional co-activation and inhibition, post-transcriptional modifications, or as decoys (Figure 1). Importantly, accumulating data from post-GWAS initiatives have demonstrated links between inherited variants in lncRNAs and cancer susceptibility.
Figure 1. Diverse cellular functions of long non-coding RNA. Source: Long non-coding RNAs: insights into functions. Tim R. Mercer, Marcel E. Dinger & John S. Mattick. Nature Reviews Genetics 10, 155-159
LncRNAs are dysregulated in many tumor tissues, including ovarian tumors. Preliminary work evaluating lncRNA expression in matched EOC tumor-normal pairs identified 99 lncRNAs that were differentially expressed (Figure 2). This preliminary list of candidate lncRNAs includes MALAT1, a bonafide lncRNA linked to numerous malignancies. MALAT1 expression was 2.6-fold higher in normal ovaries compared to ovarian tumor tissues, consistent with recent data supporting a tumor suppressor role of MALAT1 in EOC metastasis.
lncRNAs and ovarian cancer risk
Using pooled GWAS from North America, the United Kingdom and Poland, we analyzed inherited variants in 74 lncRNAs and identified several lncRNAs associated with EOC risk, including WT1-AS (Wilms’ tumor 1 antisense transcript) and HOXA11-AS (HOXA11 antisense RNA). In a functional study of EOC low risk SNP (rs17427875) in HOXA11-AS, we showed that HOXA11-AS is a tumor suppressor and that the risk allele had a greater suppressive function than the common allele (Figure 3). Furthermore, expression levels of HOXA11-AS were significantly lower in ovarian tumors than normal ovarian tissues. We also identified HOXA11 as a target gene of HOXA11-AS and showed that the minor allele negatively regulates HOXA11 more significantly than the common allele.
Figure 2. Clustergram showing 99 differentially expressed lncRNAs that distinguish between tumor & normal tissues. Low expression values are green, and high expression values are red.
Figure 3. Allele specific analyses of rs17427875 on HOXA11-AS and its targeted gene HOXA11. The minor allele of HOXA11-AS inhibited (A) cell migration, (B) invasion, and (E) tumor growth more significantly than the common allele.
Given the cumulating evidence for a functional role of genetic variation within non-coding RNA , we have developed a study (R01 CA191619) to focus specifically on long non-coding RNA in epithelial ovarian cancer (EOC) risk. Our study seeks to apply an integrative molecular epidemiologic approach to identify and characterize novel risk SNPs within lncRNA that influence EOC susceptibility. This project encompasses four main aims (Figure 4): 1) Identify lncRNA SNPs associated with EOC risk and ovarian-related lncRNAs; 2) Detect lncRNAs that show differential expression in high-grade serous EOC tumors compared to normal tissues; 3) Correlate lncRNA genotype with lncRNA expression and identify lncRNA target coding genes; and 4) Evaluate the functional role of risk SNPs and candidate lncRNAs in EOC development.
Figure 4. Comprehensive research plan to identify and functionally characterize ovarian cancer risk SNPs within lncRNA.