Specific patterns of PIWI-interacting small noncoding RNA expression in dysplastic liver nodules and hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the result of a stepwise process, often beginning with development within a cirrhotic liver of premalignant lesions, morphologically characterized by low- (LGDN) and high-grade (HGDN) dysplastic nodules. PIWI-interacting RNAs (piRNAs) are small noncoding RNAs (sncRNAs), 23-35 nucleotide-long, exerting epigenetic and post-transcriptional regulation of gene expression. Recently the PIWI-piRNA pathway, best characterized in germline cells, has been identified also in somatic tissues, including stem and cancer cells, where it influences key cellular processes.Small RNA sequencing was applied to search for liver piRNAs and to profile their expression patterns in cirrhotic nodules (CNs), LGDN, HGDN, early HCC and progressed HCC (pHCC), analyzing 55 samples (14 CN, 9 LGDN, 6 HGDN, 6 eHCC and 20 pHCC) from 17 patients, aiming at identifying possible relationships between these sncRNAs and liver carcinogenesis. We identified a 125 piRNA expression signature that characterize HCC from matched CNs, correlating also to microvascular invasion in HCC. Functional analysis of the predicted RNA targets of deregulated piRNAs indicates that these can target key signaling pathways involved in hepatocarcinogenesis and HCC progression, thereby affecting their activity. Interestingly, 24 piRNAs showed specific expression patterns in dysplastic nodules, respect to cirrhotic liver and/or pHCC.The results demonstrate that the PIWI-piRNA pathway is active in human liver, where it represents a new player in the molecular events that characterize hepatocarcinogenesis, from early stages to pHCC. Furthermore, they suggest that piRNAs might be new disease biomarkers, useful for differential diagnosis of dysplastic and neoplastic liver lesions.

Keywords: hepatocarcinogenesis; hepatocellular carcinoma; piRNAs; small non-coding RNA; smallRNA-seq.

Figure 1. piRNA expression and genomic distribution in cirrhosis and HCC

(A) Heatmap of piRNAs expression profile in human liver. (B) Catalogue of liver piRNAs identified. In the Circos plots, the outside ring shows the chromosome ideograms, with an annotation of the protein-coding transcripts in which each of them map. The middle and inner rings display average piRNA expression value in cirrhotic and HCC tissues, respectively. (C) Hierarchical clustering of piRNAs differentially expressed in matched cirrhotic vs HCC samples, identified with a non-parametric Wilcoxon Mann-Whitney test; log2(transformed RPM)-median centered expression (left) and average level of expression (right) are displayed for each RNA. Orange arrows mark piRNAs found deregulated in multiple cancers by Martinez et al. [30].

Figure 2. piRNAs-like RNA expression and genomic distribution in cirrhosis and HCC

(A) Heatmap of piRNAs-like expression profile in human liver. Only piRNAs-like with median expression > 0 in at least one sample group are included. (B) Sequence logo showing nucleotide distribution in piRNAs and piRNA-likes expressed in liver, compared to that of germline piRNAs present in piRNAbank database. (C) Catalogue of human liver piRNA-like identified. In the Circos plots, the outside ring shows the chromosome ideograms, with an annotation of the protein-coding transcripts in which each of them map. The middle and inner rings display average piRNAs-like expression value of cirrhotic and HCC tissues, respectively. (D) Hierarchical clustering of piRNAs-likes differentially expressed in matched cirrhotic vs HCC samples, identified with a non-parametric Wilcoxon Mann-Whitney test; log2(transformed RPM)-median centered expression (left) and average level of expression (right) are displayed. Mitochondrial piRNA-likes are highlighted in pink.

Figure 3. piRNA expression in early stages of hepatic carcinogenesis

Boxplots summarizing differences in expression of piRNAs and piRNA-likes between cirrhotic (CIRR), LGDN, HGDN, eHCC and HCC samples, identified with a non-parametric Wilcoxon Mann-Whitnet test. Red and blue asterisks mark significant differences (p ≤ 0.05) respect to cirrhosis and HCC, respectively.

Figure 4. Overview of piRNA expression changes during human liver carcinogenesis

The scheme summarizes the main results of piRNA and piRNA-like expression changes detected at different steps of human hepatocarcinogenesis. Deregulation of some of these molecules in dysplasia or HCC respect to cirrhosis supports the hypothesis that deregulation of these molecules may be implicated in tumor onset. A potential involvement of these small RNAs in malignant transformation and tumor progression is further suggested by specific changes in their expression confined to HGDN, eHCC and pHCC, or only in overt cancerous lesions. The green and red boxes indicate, respectively, piRNAs down- and up-regulated in HCC samples respect to paired non cancer tissues.