Characterization of hepatocarcinogenesis by transcriptome and exome sequencing via chronic HBV X expression and aflatoxin B1 exposure in immortal hepatic cell

Abstract Backgrounds: Hepatitis B virus (HBV) and aflatoxin B1 (AFB1) exposure have synergistic effects on the occurrence and progression of hepatocellular carcinoma. In this study, we constructed HBV X (HBX) expression and AFB1 exposure models promoting hepatocarcinogenesis. Methods The HBX-overexpressing HL-7702 cell model was constructed by lentivirus transfection. In vitro experiments and transcriptome sequencing determined HBX functions and transcriptional traits. An interventional system containing AFB1, NAPDH, liver microsomes and medium was constructed to mimic AFB1 exposure. Transcriptome and exome sequencing identified transcriptional traits and alterations in exon regions. Differentially expressed (differential) RNAs involved in HBX overexpression, AFB1 exposure and the AFB1 + HBX exposure groups were validated by real-time polymerase chain reaction assays. Results HBX overexpressing showed decreased capacity for cellular migration, proliferation, invasion and clonal formation compared with control cells (P ≤ 0.05). Mutational “fingerprints” of AFB1 (TP53-R249S, AGG > AGT) and novel mutations at codons 247, 249 and 250 were unveiled in the model. Transcriptome sequencing identified the following differential RNAs: 285 mRNAs, 9 miRNAs, 78 long non-coding RNAs (lncRNAs) and 117 circular RNAs (circRNAs) in HBX group; 585 mRNAs, 143 miRNAs, 208 lncRNAs and 58 circRNAs in the AFB1 group; and 564 mRNAs, 126 miRNAs, 233 lncRNAs and 61 circRNAs in the AFB1 + HBX group. Enrichment analysis suggested the involvement of differential RNAs in angiogenesis, oxidative phosphorylation and ribosome pathways, etc. Additionally, competing endogenous RNA networks of differential mRNA-miRNA-lncRNA/circRNA cross-talk were constructed in the above groups. Exome sequencing unveiled 1061 (AFB1) and 3601 (AFB1 + HBX) single nucleotide variations, 30 (AFB1) and 40 (AFB1 + HBX) insertion and deletion alterations, 1068 (AFB1) and 138719 (AFB1 + HBX) copy number variations. The number of alterations was consistently higher in the AFB1 + HBX group compared to the AFB1 group. Conclusions We successfully constructed a cellular model mimicking HBX expression, AFB1 exposure and provided novel insights for understanding AFB1/HBX-related hepatocarcinogenesis.