The experimental study of genomic alterations of hepatocellular carcinoma by panel-based sequencing

Abstract

Objective To explore the value of high-throughput capture sequencing technology of tumor gene in detection of somatic mutation and mutation patterns of hepatocellular carcinoma cells. Methods Based on the target gene sequencing technology of the second generation sequencing technology, a custom sequence capture system containing 325 tumor related genes panel, NimbleGen, was used, and the target region of 7 hepatoma cell lines (HuH-7, Hep3B, SK-HEP-1, MHCC97H, MHCC97L, HepG2, HepG2.2.15) DNA gene sequencing were captured and sequenced. The tumor-related mutant gene and mutation load rate, as well as the difference between the gene mutation trend were analysed. Results The results showed that the average coverage rate of the target area was 99.7%, and the average sequencing depth was about 1 000×, and 100 genes(mutation frequency≥5%)were detected, including 344 non synonymous mutations. There are 38 gene mutations closely related to cancer enriching in 5 oncogenic signaling pathways: chromatin remodeling, Notch, mitogen-activated protein kinase (MAPK), p53, and Wnt/beta-catenin pathways. Compared with the previous reports of somatic mutations in hepatocellular carcinoma (HCC) patients, the chromatin remodeling and gene mutations in the Notch signaling pathway were higher in HCC cell lines. Three mutant genes not reported in HCC patients ever were identified, including two tumor suppressor genes [MSH6, sex determining region Y box 9 (SOX9)] and one oncogene (ALK).44 mutant genes were identified in MHCC97 cell lines (MHCC97H and MHCC97L), of which 30 (68%) were co-mutations. Mutation genes are enriched in oxidative stress pathways (KEAP1), chromatin remodeling (NCOR1, KMT2D, ARID1A), MAPK (MAP3K1, MAP2K3) and Wnt/beta-catenin (AXIN1, SOX9) pathways. ALK and ERBB2 mutations were detected only in MHCC97L, APOB mutations only in MHCC97H. Notch pathway (Notch1, Notch2) had higher mutation frequency in MHCC97L, TERT in MHCC97H.37 mutant genes were identified in HepG2 and HepG2.2.15, of which 19 (51%) were co-mutations. HepG2 and HepG2.2.15 had the same mutation sites and similar mutation frequency in Notch pathway (Notch1, Notch2) and chromatin remodeling pathway (KMT2D, ZFHX4). Tp53 mutations were detected only in HepG2, adenomatous polyposis coli (APC) and ataxia-telangiectasia mutated (ATM) mutations only in HepG2.2.15. The four HCC cell lines (HuH-7, Hep3B, SK-HEP-1, HepG2) from different HCC patients had different mutation modes, but the same clone derived HCC cell lines showed a large similarity. Conclusion High throughput sequencing technology of tumor gene has high application value in the detection of cell lines and mutation patterns in hepatocellular carcinoma cell lines. Key words: Hepatocellular carcinoma; Gene mutation; Panel-based Sequencing