Suppression of Noncoding RNAs as Shared Early Genetic Events in Multistep Hepatocarcinogenesis
Authors: Takeshi Suda, Seiichi Yoshikawa, Tsutomu Kanefuji, Satoshi Abe, Takahiro Hoshi, Shinichi Morita, Kazuyoshi Yagi, Shuji Terai
Journal: Hepatology (2021)

Common genetic traits are not well defined for HCC because long-lasting necroinflammation facilitates various genetic errors in hepatocytes prior to hepatocarcinogenesis. A 73-year-old Japanese female who had chronic hepatitis C for decades was referred to our hospital due to multiple liver tumors. Contrast-enhanced MRI with gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid classified the tumors into three types based on the combinations of signal intensities in specific sequences and dynamic phases (Fig. 1). One of each type of tumor and surrounding nontumorous (NT) liver tissue was obtained under ultrasound guidance. Histological diagnoses of types 1, 2, and 3 revealed suspicion of dysplastic nodules (DNs), well-differentiated HCC (WEL), and moderately differentiated HCC (MOD), respectively. Biotinylated sense-strand complementary DNA was generated and hybridized to GeneChip Human Transcriptome Array 2.0 (Affymetrix, Santa Clara, CA). The raw data of 913,605 signals covering 67,528 genes were collected for each sample and deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GSE 153565) after normalization. In total, 2,855 genes showed at least a 2-fold increase and 3,206 at least a 2-fold decrease in expression in tumor samples compared to NT samples; 57.7%, 12.4%, and 3.9% were specific for MOD, WEL, and DN, respectively (Fig. 2A; transcriptome analysis console 4.0.1). Only 7.3% were up-regulated or down-regulated and shared among all tumor types. Down-regulation was more frequent among these shared genes than among those with expression changes specific for each tumor type (P < 0.0001, Fisher's exact test with Bonferroni correction). The dysregulated gene set shared in all tumor types mostly consisted of immune-related genes, noncoding genes including 94 small nucleolar RNAs, or genes with unknown function, with frequencies of 19.7%, 24.4%, or 50.5%, respectively (Supporting Table S1). The principal component analysis (PCA) incorporating all genes for distinguishing tumors from NT defined three dimensions of PCA1 to PCA3, in which MOD, WEL, and DN were radially plotted from NT tissue along almost each dimension (Fig. 2B). Hierarchical clustering of typical genes determining PCA1, PCA2, or PCA3 discriminated MOD, WEL, or NT from others (Fig. 2C). Functional targets of typical genes include xenobiotics, immunity, and anion transport for PCA1; energy metabolism and complement for PCA2; and noncoding RNAs (ncRNAs) for PCA3. Information for common genetic codes causing each disorder potentiates the development of a specific molecular targeting therapy such as imatinib mesylate for chronic myeloid leukemia. Unfortunately, evaluation of classical HCCs from various patients reveals large genetic variations.(1) Actually, a path from NT tissue through DN and WEL to MOD for each tumor in our case was not observed by PCA; instead, the expression profiles of each tumor deviated from NT tissue along each axis, suggesting that multicentric HCCs may not dedifferentiate by following steps in a common pathway. Overall, minimizing genetic variations unconnected to carcinogenesis should be critical to elucidate a common genetic event in hepatocarcinogenesis. Comparison of numerous expression profiles from DN, WEL, and MOD with consecutive evaluation of a single liver from the very beginning of hepatocarcinogenesis would be the least biased approach. Recently, pan-cancer analysis of whole genomes revealed that 25% of cancers carry at least one putative noncoding driver mutation.(2) Consistently, this brief report suggests that approximately one-fourth of expression changes shared with all types of tumors involve ncRNAs, which were major constituents of PCA3. The ncRNAs comprise >90% of the RNAs made from the human genome and play a critical role in cancer. Because small nucleolar RNAs were the most abundant dysregulated ncRNAs that were shared in all tumor types, they were further annotated using the web-based database snoRNA in Cancers (http://bioinfo.life.hust.edu.cn/SNORic).(3) Consequently, several small nucleolar RNAs revealed consistent results of significant repression in HCCs irrespective of histologic grades and pathologic stages (Supporting Fig. S1). The results suggest that errors suppressing expression of noncoding regions may play a key role in the early stage of hepatocarcinogenesis. Although observations in one case may result in an inadequate assessment of biological variabilities and should be confirmed in a larger cohort, molecular targeting of these ncRNAs has the potential to pave a new path for preventing HCC development by overcoming the current limitation of targeted therapies for HCCs at advanced stages, at which genetic traits are highly heterogenous.(1) Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.