Exon 2 deletion splice variant of γ-glutamyl carboxylase causes des-γ-carboxy prothrombin production in hepatocellular carcinoma cell lines

Abstract 

Using GGCX gene-specific real-time PCR, exon 2 deletion splice variant of vitamin K-dependent γ-glutamyl carboxylase (GGCX) mRNA was identified in HCC cell lines. Expressions of wild type and exon 2 deletion variant of GGCX were analyzed with relevance to DCP production in HCC cell lines. Hep3B, HepG2, HuH1, HuH7, and PLC/PRF/5 produced DCP, while SK-Hep-1, HLE, HLF, and JHH1 produced no detectable level of DCP. DCP-producing cells expressed exon 2 deletion variant of GGCX mRNA and protein, while DCP-negative cells expressed no detectable level of exon 2 deletion variant of GGCX. These results suggest that exon 2 deletion splice variant of GGCX causes dysfunction of GGCX enzyme activity resulting in DCP production in HCC cell lines.

Keywords: Tumor markers, Splice variant, Vitamin K-dependent proteins, Dominant-negative inhibitor, Prothrombin

Abbreviations: DCP, des-γ-carboxy prothrombin, HCC, hepatocellular carcinoma, GGCX, vitamin K-dependent γ-glutamyl carboxylase, AFP, α-fetoprotein, VKOR, vitamin K epoxide reductase, Δ2GGCX, exon 2 deletion splice variant of GGCX, PLC, PLC/PRF/5, DMEM, Dulbecco's modified Eagle's medium, FBS, fetal bovine serum, ECLIA, electrochemiluminescence immunoassay, TBS-T, TBS with Tween-20, WT, wild type, PCR, polymerase chain reaction, RT-PCR, real-time quantitative PCR, ACTB, β-actin, CMVp, cytomegalovirus promoter, GFP, green fluorescent protein, ESE, exonic splicing enhancer