PRJNA371753: RNA sequencing (RNA-SEQ) of 21 HBV-HCC patients with non-neoplastic liver and tumor tissues

Summary: Purpose: Chronic Hepatitis B virus (HBV) infection leads to liver fibrosis which is a major risk factor in Hepatocellular carcinoma (HCC) and an independent risk factor of recurrence after HCC tumor resection. HBV genome can be inserted into human genome, and chronic inflammation may trigger somatic mutations. Several studies characterized HBV integration sites in HCC patients with regard to frequently occurring hotspots. However, how HBV integration and other genomic changes contribute to the risk of tumor recurrence with regard to different degree of liver fibrosis is not clearly understood. In this study, we aim to find potential molecular mechanisms underlying tumor recurrence of HBV-associated HCC (HBV-HCC) with different degree of liver fibrosis. Methods: We performed RNA sequencing of 21 pairs of tumor and non-neoplastic liver tissues of HBV-HCC patients and performed comprehensive genomic analysis of our RNAseq data and public available sequencing data related to HBV-HCC. We developed a robust pipeline for sensitively identifying HBV integration sites based on sequencing data. Simulations with sequencing data showed that our method outperformed existing methods. We also compared SNPs of each sample with SNPs in cancer census database and inferred patient’s pathogenic SNP loads in tumor and non-neoplastic liver tissues. Conclusions: The HBV-integration and pathogenic SNP load patterns for HCC recurrence risk vary depending on liver fibrosis stage, suggesting potentially different tumorigenesis mechanisms for low and high liver fibrosis patients.

Overall Design: Total RNA (1-3μg/sample) extracted from surgical resection specimens were submitted to the Mount Sinai Genomic Core Facility for quality control analysis. The RNA quality was assessed using the Agilent 2100 Bioanalyzer, and the RNA integrity number for all 21 samples measured 8.2 ± 0.7 (mean ± SD). The poly(A)-RNA was captured using oligo-dT beads and used for cDNA library preparation using the standard TruSeq RNA Sample Prep Kit v2 protocol (Illumina, CA, USA). Briefly, total RNA was poly-A selected and then fragmented. The cDNA was synthesized using random hexamers, end-repaired and ligated with appropriate adaptors for sequencing. The library then underwent size selection and purification using AMPure XP beads (Beckman Coulter, CA, USA). The appropriate Illumina recommended 6 bp barcode bases are introduced at one end of the adaptors during PCR amplification step. The size and concentration of the RNAseq libraried was measured by Bioanalyzer and Oubit fluorometry (Life Technologies, NY, USA) before loading onto the sequencer. The mRNA libraries were sequenced on the Illumina HiSeq 2500 System with 100 nucleotide paired-end reads, according to the standard manufacturer’s protocol (Illumina, CA, USA).