Alternative Splicing In The Hepatocellular Carcinoma:a Transcriptome Analysis With The RNA Sequencing

Alternative splicing plays an important role in regulation of gene expression and it is the main mechanism to produce protein diversity. Alternative splicing has been related to a variety of tumors including hepatocellular carcinoma (HCC).In this work, we aim to study the role of alternative splicing in HCC progress and development by comparison patterns of alternative splicing between HCC and normal tissues using the second generation sequencing technology. For the current work, we sampled three tumor sections and three adjacent normal tissues from an HCC patient. Whole transcriptome sequencing and bioinformatics analysis of alternative splicing were conducted on these6samples. The resuts showed that there are2061genes with significant differences in alternative splicing between tumor and normal tissues, of which661genes are with different promoter use and1400genes are with different alternative splicing, but sharing transcription start site. Also, we found that4149isoforms show significant up regulation while3539isoforms show significant down regulation in HCC. Isoform diversity in tumor and normal tissues at lowly and medially expressed genes are at the same level. However, as to highly expressed genes, isoform diversity is higher in tumor tissues. This is the first time alternative splicing and isoform diversity are used together to describe the differences of transcriptional level between HCC and normal tissues in the studies of alternative splicing.We carried out GO functional enrichment analysis and KEGG signaling pathway enrichment analysis on these differentially spliced genes, we found that some of them are related to cell death and are enriched in several tumor signaling pathways. Genes with differential alternative splicing are mostly in the upstream of signaling pathways while genes of different expression are likely to be the downstream targets. For example, genes in the Wnt signaling pathway such as MYC, JUN and CCND1, which are related to cell cycle and apoptosis, are differentially expressed. These may largely due to the differences in alternative splicing of APC, CTNNB3and transcription factor TCF/LEF. This suggests that alternative splicing can be associated with HCC through activation of certain tumor signaling pathways by affecting the expression of genes related to cell cycle and apoptosis. So we studied the way alternative splicing acts on HCC from a whole view rather than one or two genes.Meanwhile, we noted that a portion of the up regulated genes in HCC are related to spliceosome and splicing regulation factors SR protein and hnRNP protein. This is likely to be the reason for the differences in alternative splicing between tumor and normal tissues.Lastly, we found that some small nucleolar RNAs are specifically highly expressed in HCC when we try to analyze tumor and normal tissue specific expressed isoforms. This is a new discovery. Based on the current research, we propose a hypothesis that these small nucleolar RNAs may play an important role in HCC initiation and progression.