Effect of carboxyl-terminal truncated HBx mediated down-regulation of RhoGDIα expression on the metastasis and invasion of hepatocellular carcinoma

Abstract

Objective To investigate the mechanism of natural carboxyl-terminal truncated HBx (with 31 amino acids deleted at the C-terminal end) (HBxΔ31)-dependent down-regulation of Rho GDP dissociation inhibitor alpha (RhoGDIα) expression and its role in enhancing hepatocellular carcinoma (HCC) metastasis. Methods HepG2 cells with stable expression of wild type HBx and its deletion mutant HBxΔ31 protein were selected as the study subjects. The effects of HBx and HBxΔ31 on RhoGDIα expression in HepG2 cells was detected using real-time quantitative reverse transcription PCR (RT-qPCR) and Western blot analysis. A series of deleted and mutated variants of the RhoGDIα promoter were made and individually co-transfected with HBx-and HBxΔ31-expressing vectors or control empty vector into HepG2 cells. The luciferase reporter assay was used to identify the cis-regulatory elements of the RhoGDIα promoter in response to HBxΔ31 regulation. The interaction between Myc-associated zinc finger protein (MAZ) and HBxΔ31 was examined by co-immunoprecipitation experiment. Eelectrophoretic mobility shift assay (EMSA) was performed to determine interaction of MAZ with the RhoGDIα promoter in HBxΔ31-expressing HepG2 cells. The impact of reduced RhoGDIα expression by HBxΔ31 on cell-invasive activity was analyzed by the Matrigel cell invasion assay. In addition, the effects of silencing of shRNA-mediated RhoGDIα in HepG2 or introduction of RhoGDIα by transfection in HBxΔ31-expressing HepG2 cells on cell-invasion were investigated. Results HBxΔ31, but not HBx, suppressed RhoGDIα expression at transcriptional levels. Analysis of the deletion and mutation of RhoGDIα promoter showed that the HBxΔ31 repressive element localized between nt-460 and-242 bp of RhoGDIα promoter and that the transcription factor MAZ binding sites was required for RhoGDIα promoter inactivation regulated by HBxΔ31. In addition, HBxΔ31 represses RhoGDIα expression by enhancing MAZ binding to its promoter through directly associating with MAZ. The cell-migratory and cell-invasive activity were significantly increased in sh-RhoGDIα-expressing HepG2 cells, as compared to control cells (migrated cells number: 58 ± 5 vs. 98 ± 7, invaded cells number: 55 ± 6 vs. 113 ± 6, t= 18.91 and t= 20.12, both P < 0.01). However, ectopic expression of RhoGDIα in HBxΔ31-expressing HepG2 cells significantly decreased cell migration and invasion (migrated cells number: 40 ± 4 vs. 115 ± 5, invaded cells number: 42 ± 4 vs. 102 ± 4, t= 18.14 and t= 16.31, both P < 0.001). Conclusion Carboxyl-terminal truncated HBx deregulates RhoGDIα expression through MAZ, in turn which promotes the invasion and metastasis of HCC. Key words: Carcinoma, hepatocellular; Sequence deletion; Hepatitis B virus; Gnanine nucleotide dissociation inhibitors; Neoplasm metastasis