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Background Integration of HBV DNA into the human genome could progressively contribute to hepatocarcinogenesis. Both intrahepatic cholangiocarcinoma (ICC) and combined hepatocellular-cholangiocarcinoma (CHC) are known to be associated with HBV infection. However, the integration of HBV and mechanism of HBV-induced carcinogenesis in ICC and CHC remains unclear. Methods 41 patients with ICC and 20 patients with CHC were recruited in the study. We conducted HIVID analysis on these 61 samples to identify HBV integration sites in both the tumor tissues and adjacent non-tumor liver tissues. To further explore the effect of HBV integration on gene alteration, we selected paired tumors and adjacent non-tumor liver tissues from 3 ICC and 4 CHC patients for RNA-seq and WGS. Results We detected 493 HBV integration sites in ICC patients, of which 417 were from tumor samples and 76 were from non-tumor samples. And 246 HBV integration sites were detected in CHC patients, of which 156 were located in the genome of tumor samples and 90 were in non-tumor samples. Recurrent HBV integration events were detected in ICC including TERT, ZMAT4, MET, ANKFN1, PLXNB2, and in CHC like TERT, ALKBH5. Together with our established data of HBV-infected hepatocellular carcinoma, we found that HBV preferentially integrates into the specific regions which may affect the gene expression and regulation in cells and involved in carcinogenesis. We further performed genomic and transcriptomic sequencing of three ICC and four CHC patients, and found that HBV fragments could integrate near some important oncogene like TERT, causing large-scale genome variations on nearby genomic sequences, and at the same time changing the expression level of the oncogenes. Conclusion Comparative analysis demonstrates numerous newly discovered mutational events in ICC and CHC resulting from HBV insertions in the host genome. Our study provides an in-depth biological and clinical insights into HBV-induced ICC and CHC.

Background and Aims: Cholangiocarcinoma (CCA), the second most common hepatobiliary cancer, is associated with poor prognosis. Therefore, there is a need to elucidate on the pathogenic mechanisms of CCA. In this study, we aimed at identifying lncRNA-related prognostic signatures for CCA through bioinformatics analysis and further validated their functions in CCA tumorigenesis and progression.Methods: The RNA-seq data of CCA were downloaded from public databases. Differentially expressed lncRNAs (DElncRNAs) were screened using R packages. Then, candidate OS- and DFS-related DElncRNAs were selected through Kaplan–Meier survival analysis. Furthermore, LASSO regression analyses were performed to establish two prognostic signatures, termed the OS and DFS signatures, respectively. Multivariate COX models and nomograms for overall survival (OS) and disease-free survival (DFS) were established based on OS/DFS signature and clinical data. Hub lncRNAs were identified and enrichment analyses performed to explore their potential functions. Finally, in vitro and in vivo models were used to validate the effects of the hub lncRNAs in CCA tumorigenesis and progression.Results: A total of 925 DElncRNAs were selected, from which six candidate OS-related lncRNAs and 15 candidate DFS-related lncRNAs were identified. The OS and DFS signatures were then established using four lncRNAs, respectively. We found that the OS signature and vascular invasion were significant independent risk factors for OS outcomes of CCA, while the DFS signature, vascular invasion and CA19-9 were significant independent risk factors for DFS outcomes of CCA. MiR4435-2HG and GAPLINC were selected as hub lncRNAs because they were included in both OS and DFS signatures. GO and KEGG enrichment analyses revealed that the two hub lncRNAs were involved in CCA tumorigenesis and progression. Finally, we constructed in vitro and in vivo models and revealed that the lncRNAs, MiR4435-2HG and GAPLINC can prompt CCA proliferation and migration in vitro and in vivo.Conclusions: The established OS and DFS signatures, which were based on DElncRNAs, are independent risk factors for OS and DFS of CCA patients, respectively. MIR4435-2HG and GAPLINC were identified as hub lncRNAs. In vitro and in vivo models revealed that MiR4435-2HG and GAPLINC can prompt CCA progression, which might be novel prognostic biomarkers and therapeutic targets for CCA.