The Study Of Human Hepatocellular Carcinomas Revealed By Whole-exome And Transcriptome Sequencing

Background and Objective:Primary liver cancer (PLC) is the eighth most common cancer, with an estimated annual incidence of1million and mortality of500,000cases worldwide. As the most common type of PLC, hepatocellular carcinoma (HCC) represents about80-90%of the primary malignant tumors of the liver observed in adults. Patients who develop HCC have the remarkably poor prognosis, with8.9%survival rate at5years in the United States. Etiology research has identified several major HCC-associated factors, such as chronic hepatitis B and C viral infection, alcohol consumption, aflatoxin-B1-contaminated food, and hemochromatosis; over50%of HCC cases worldwide are attributable to HBV infection. In the past few decades, many studies have been done to define the molecular mechanisms driving hepatocarcinogenesis. Several genes have shown involvement in genetic and epigenetic events in HCC, including tumor suppressor genes (TSG)(TP53, p16, and p21), β-catenin, ErbB family, COX-2, and HGF, et al. In addition, HCC also displays gross genomic alterations, including chromosomal instability (CIN), CpG island methylation. DNA rearrangements, nucleotide substitution, and microsatellite instability (MSI). Among them, the acquisition of somatic genetic aberrations has proven to be the driving forces of tumor development and progression, which include single base substitutions, translocations and copy-number variations. Systematic efforts have been made to characterize such alterations associated with several human cancer types by cytogenetic studies and array-based profiling.Next generation sequencing (NGS) technology would provide extensive and detailed information about specific genetic alterations within a tumor, which can produce an enormous volume of data at a drastically reduced the cost. This opens the door for carrying out very detailed genetic analyses of cancer tissue, potential with it becoming a standard approach for tumor analysis. The recent announcement of complete genome sequencing of AML using Illumina/Solexa sequencing technology highlighted the potential of NGS gleaning rich variant information for advanced tumor analysis.Here, we used NGS technology to carry out whole-exome and transcriptome sequencing for liver cancer tissues. Methods:1. We carried out whole-exome and transcriptome sequencing for nine paired tumor and adjacent non-tumor tissues, and then screened the data.2. We validated the whole-exome and transcriptome sequencing results by qRT-PCR, western blot and immunohistochemisty.3. A total of137surgically resected liver specimens were subjected to qRT-PCR by primers of11q13.2-3and19q13.2.4. According to transcriptome sequencing data, we found a novel fusion gene in11q13.2-3named as PPFIA1-SHANK2, which was then obtained by5’&3’-RACE amplification and some functional study was also performed.5. We campared affymetrix SNP6.0array with whole-exome sequencing to detect CNVs.6. We validated some transcriptome sequencing data.Result:A high-resolution Copy-number variations (CNVs) screening approach was performed in nine pairs hepatocellular carcinoma(HCC) by intensive whole-exome and transcriptome sequencing. We identified62262flag exons encompassing415single and136multi-gene CNVs,26.6%and41.9%of which were successfully verified totally (versus the approximate6%positive events via array-based methods), including the high frequent duplication of11q13.2-13.3reported previously. Importantly, the novel somatic amplification of19q13.2in tumor tissues was experimentally verified in-26%and even73%HCC individuals as compared to their non-tumor adjacent tissues and their counterpart blood specimens, respectively. Correlation and survival analysis further indicated that the19q13.2amplification was significantly correlated with worse clinicopathological characteristics and OS/TTR (overall survival/time to recurrence) rates. Simultaneously, a novel fusion gene PPFIA1-SHANK2was detected by whole-exome and transcriptome sequencing on both levels of genomic DNA and mRNA respectively. And then, the full-length of this fusion gene was obtained by rapid amplification of cDNA ends (RACE). Some other transcriptome sequencing dates were also validated by qRT-PCR, western blot and immunohistochemisty.Conclusion: We detected two important CNVs including11q13.2-13.3and19q13.2through whole-exome sequencing. Importantly, the novel somatic amplification of19q13.2in tumor tissues was experimentally verified in-26%and even73%HCC individuals as compared to their non-tumor adjacent tissues and their counterpart blood specimens, respectively. Correlation and survival analysis further indicated that the19q13.2amplification was significantly correlated with worse clinicopathological characteristics and OS/TTR (overall survival/time to recurrence) rates. Simultaneously, a novel fusion gene PPFIA1-SHANK2was detected by whole-exome and transcriptome sequencing on both levels of genomic DNA and mRNA respectively. And then, the full-length of this fusion gene was obtained by rapid amplification of cDNA ends (RACE). And functional study show that it could promote cell migration and invasion.