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. 2016 Jul 4;7(34):55110–55127. doi: 10.18632/oncotarget.10399

Figure 1. Detection of full-length and C-terminal truncated forms of HBx DNA in human HCC samples and mRNA in human hepatoma cell lines.

Figure 1

A. Representative results showed the presence of C-terminal truncated and full-length HBx DNA in HCC tumors (T) and their corresponding non-tumorous liver tissues (N). Case 135 had C-terminal truncated HBx DNA in tumor and full-length HBx in non-tumorous tissue. Case 289 showed the presence of full-length and C-terminal truncated HBx in both tumor and non-tumorous tissue, whereas case 305 was from an HBsAg-negative patient and showed no HBx DNA in both tumor and non-tumorous liver tissue. B. Sequencing analysis of PCR products showed the presence of two specific substantial deletions within HBx DNA from HCC tissues. On the upper panel: sequence alignment of full-length HBx DNA from a control sequence of HBV adw2 subtypes (GenBank accession number X02763) and case 135 with the deletion at nt 367-374 (codons 123-125), and case 210 with the deletion at nt 370-377 (codons 124-126). The 8-nucleotide deletions are shown as “////////”. Lower letter represents the substantial nucleotide. On the lower panel: alignment of deduced amino acids (aa) at C-terminal end of HBx DNA from a control sequence of HBV adw2 (154aa) and HCC tissues of two patients (cases 135 and 210). Case 135: deletion at nt 367-374, frame shift and new stop codon formation, resulting in loss of 34aa and formation of 3 new amino acids. Case 210: deletion at nt 370-377, frame shift and new stop codon formation, resulting in loss of 33aa and formation of 2 new amino acids. The expected HBx protein of cases 135 and 210 each would be 31aa shorter than full-length HBx protein, denoted as HBxΔ31. Solid square represents new stop codon formed due to deletion and frame shift in HBx DNA. Lower letter represents the new amino acids. C. Binding locations of two PCR primer pairs targeting the full-length (F1-R5) and 3′-deleted (F1-R1) HBx gene. D. One representative example of HCC cases with HBx truncation in tumors, wherein the short HBx fragment were amplified from both tumor (T) and non-tumorous (N) tissues, but full-length HBx fragment was amplified only from adjacent non-tumorous liver tissues. β-actin was used as an internal control. E. RT-PCR analysis in HCC cell lines showed the presence of HBxΔ31 transcripts only in MHCC-LM3 cells, but not in other HCC cell lines or the immortalized normal liver cell lines.