Transcriptome Analysis Of HCC By Next-generation Sequencing

RNA-seq is a powerful tool for comprehensive characterization of whole transcriptome at both gene and exon levels and with a unique ability of identifying novel splicing variants. To date, RNA-seq analysis of HBV-related hepatocellular carcinoma (HCC) has not been reported. In this study, we performed transcriptome analyses for 10 matched pairs of cancer and non-cancerous tissues from Chinese patients with HBV-related HCC using 36bp single-end sequencing approach on Solexa/Illumina GAII platform. On average, about 21.6 million sequencing reads and 10.6 million aligned reads were obtained for samples sequenced on each lane, which was able to identify > 50% of all the annotated genes for each sample. Furthermore, from by far the largest database of transcripts expressed in HCC tissues, we identified 1,378 significantly differently expressed genes (DEGs) and 24,338 differentially expressed exons (DEEs). Comprehensive function analyses indicated that cell growth-related, metabolism-related and immune-related pathways were most significantly enriched by DEGs, pointing to a complex mechanism for HCC carcinogenesis. Positional gene enrichment analysis showed that DEGs were most significantly enriched at chromosome 8q21.3-24.3. The most interesting findings were from the analysis at exon levels where we characterized three major patterns of expression changes between gene levels and exon levels, implying a much complex landscape of transcript-specific differential expressions in HCC. Finally, we identified a novel highly up-regulated exon-exon junction in ATAD2 gene in HCC tissues. Overall, to our best knowledge, our study represents the most comprehensive characterization of the HBV-related HCC transcriptome including exon level expression changes and novel splicing variants, which illustrated the power of RNA-seq and provided important clues for understanding the molecular mechanisms of HCC pathogenesis at system-wide levels.The key study contents:(1) evaluation of data quality and sequencing depth required for transcriptome analysis.(2) Comparison between RNA-seq and microarray.(3) The analysis and validation of differential gene expression. (4) The chromosome locations of DEGs.(5) Functional annotation of DEGs.(6) Exon expression level analysis.(7) Identification of novel DE exon-exon junctions.The key study results:(1) For the first time the RNA-seq analysis of HBV-related HCC.(2) 1,378 significantly DEGs and 24, 338 DEEs were identified.(3) 54 bio-function terms and 41 canonical pathways related to HCC.(4) Many of chromosomal aberrations were identified, especially 8q24.(5) Some splicing patterns were identified.(6) A novel-splicing variant in ATAD2 was identified.Conclusion:Overall, by RNA-seq our study represents the most comprehensive characterization of the HBV-related HCC transcriptome including gene level expression changes, exon level expression changes and novel splicing variants, which provided important clues for understanding the molecular mechanisms of HCC pathogenesis at system-wide levels.