New insights into potential biomarkers and their roles in biological processes associated with hepatitis C-related liver cirrhosis by hepatic RNA-seq-based transcriptome profiling

Abstract

Chronic hepatitis C virus infection is a major cause of mortality due to liver cirrhosis globally. Despite the advances in recent therapeutic strategies, there is yet a high burden of HCV-related cirrhosis worldwide concerning low coverage of newly developed antiviral therapies, insufficient validity of the current diagnostic methods for cirrhosis, and incomplete understanding of the pathogenesis in this stage of liver disease. Hence we aimed to clarify the molecular events in HCV-related cirrhosis and identify a liver-specific gene signature to potentially improve diagnosis and prognosis of the disease.

Through RNA-seq transcriptome profiling of liver samples of Iranian patients with HCV-related cirrhosis, the differentially expressed genes (DEGs) were identified and subjected to functional annotation including biological process (BP) and molecular function (MF) analysis and also KEGG pathway enrichment analysis. Furthermore, the validation of RNA-seq data was investigated for seven candidate genes using qRT-PCR. Moreover, the diagnostic and prognostic power of validated DEGs were analyzed in both forms of individual DEG and combined biomarkers through receiver operating characteristic (ROC) analysis. Finally, we explored the pair-wise correlation of these six validated DEGs in a new approach.

We identified 838 significant DEGs (padj ˂0.05) enriching 375 and 15 significant terms subjected to BP and MF, respectively (false discovery rate ˂ 0.01) and 46 significant pathways (p-value ˂ 0.05). Most of these biological processes and pathways were related to inflammation, immune responses, and cellular processes participating somewhat in the pathogenesis of liver disease. Interestingly, some neurological-associated genes and pathways were involved in HCV cirrhosis-related neuropsychiatric disorders. Out of seven candidate genes, six DEGs, including inflammation-related genes ISLR, LTB, ZAP70, KLRB1, and neuronal-related genes MOXD1 and Slitrk3 were significantly confirmed by qRT-PCR. There was a close agreement in the expression change results between RNA-seq and qRT-PCR for our candidate genes except for SAA2-SAA4 (P= 0.8). High validity and reproducibility of six novel DEGs as diagnostic and prognostic biomarkers were observed. We also found several pair-wise correlations between validated DEGs.

Our findings indicate that the six genes LTB, ZAP70, KLRB1, ISLR, MOXD1, and Slitrk3 could stand as promising biomarkers for diagnosing of HCV-related cirrhosis. However, further studies are recommended to validate the diagnostic potential of these biomarkers and evaluate their capability as targets for the prevention and treatment of cirrhosis disease.