HBV CCNE1 fusion transcript promotes cell proliferation in hepatocellular carcinoma

Abstract

Abstract of thesis entitled

HBV CCNE1 fusion transcript promotes cell proliferation in hepatocellular carcinoma

Submitted by TUNG LAI NAR

For the degree of Doctor of Philosophy at the University of Hong Kong in January 2020

Hepatocellular carcinoma (HCC) is the sixth most common cancer in the world. Hepatitis B virus (HBV) is the most risk factor of HCC and patients with increased number of HBV integrations is associated with shorter survival time. Recurrent HBV integration into the cyclin E1 (CCNE1) genomic DNA of four patients (P268, P106, P145, P200) was found from the previous next generation sequencing study. In this study, we validated the integration events and mapped the insertion breakpoints in these integration regions. Sanger sequencing on PCR products confirmed the presence of HBV integrations existed in the DNA fragments of the tumor tissues. CCNE1 transcripts were detected through RT-PCR in the tumor (T) tissues, but no detectable PCR product was found in non-tumor (NT) tissues. It suggested up-regulation of CCNE1 transcript upon HBV integration. To compare tumor tissues of HCC patients with and without HBV integration through qPCR, the four patients’ tumors showed at least 14 folds of CCNE1 expression; while in an independent cohort of patients without HBV integration (n=116) more than 10 folds change was only 27.6%. Western blot analysis demonstrated obvious expression of CCNE1 protein in the tumor tissues compared to the corresponding non-tumor tissues of the four patients. In immunohistochemistry analysis, strong nuclear expression of CCNE1 was visualized in the tumor sections of the four patients; patients without HBV integration in CCNE1 gene showed varied CCNE1 expression. The finding of enhancer activity assay demonstrated that HBV inserts (P145 and P106) significantly increased the enhancer activity of the original genomic DNA fragment. To further investigate the promoter function in the two HBV inserts, lunciferase reporter of the pGL3-Basic vector was used. The findings suggested that the promoter and enhancer functions of the HBV insert in P145 and P106. To test the probability of the promoter activity of HBV insert driving the expression of HBx-CCNE1 chimeric transcript, RT-PCR amplification was performed on the cDNA using primers flanking the first 19bp of HBx coding region and exon 12 of CCNE1. Sanger sequencing performed on PCR products and the result showed that HBx coding region fused to CCNE1 exon 3 in P145 or to exon 5 in P106, which confirmed the expression of HBx-CCNE1 chimeric transcripts. The effect of CCNE1 expression on HCC cells was studied through both siRNA (in PLC and Hep3B cells) and shRNA (in PLC cells) mediated gene silencing approach. CCNE1 knock-down transfected cells inhibited cell proliferation, colony formation, and cell migration. To imitate tumor tissues of the patients with HBV integration, we overexpressed CCNE1 (001) and HBV fusion transcript (T145) in 97L and 97H cells. Up-regulation of HBV fusion transcript (T145) promoted cell proliferation, colony formation, and cell migration, but up-regulation of CCNE1 (001) was not sufficient to drive the expression. To conclude, the integration events in the four patients (P268, P145, P106, and P200) were validated. Novel HBV CCNE1 fusion transcript (P145 and P106) were generated. Up-regulated expression level of fusion transcript (T145) promoted cell proliferation in vitro and in vivo.

(words count = 494 words)