Hepatitis B Virus X Protein Upregulates Expression Of Capn4 To Promote Migration Of Hepatoma Cells

BACKGROUND Hepatitis B virus (HBV) infection is closely correlated with the development of hepatocellular carcinoma (HCC), in which hepatitis B virus X protein (HBx) plays crucial roles. As a protein with multifunction, HBx has been shown to be involved in transactivation, signal transduction, apoptosis, proliferation, invasion and metastasis of HCC. Although researchers have paid much closer attention to the study of HBx enhanced invasion and metastasis of HCC, the molecular mechanism of HBx enhanced invasion and metastasis is still uncertain. Newly studies reported that the expression of Calpain small subunit 1 (Capn4) is increased in local recurrence and metastasis after liver transplantation. It indicated Capn4 played an important role in invation and metastasis of HCC cells. However, whether HBx could induce the expression of Capn4 in promotion of HCC cells metastasis and the molecular mechanism of HBx induced expression of Capn4 are still not clear.OBJECTIVE This study is performed to detect the effect of HBx on Capn4 expression in HCC cells with transient or stable HBV X gene transfected, observe the role of Capn4 in HBx induced migration of HCC cells, and find out the molecular mechanism of HBx induced expression of Capn4. Then, we demonstrate a new molecular mechanism of HBx induced invasion and metastasis in HCC cells.METHODS 1. Established cell model, HepG2-X and H7402-X, in which HBx could express stably. And then we detected the differences of Capn4 expression in HepG2 (H7402) and HepG2-X (H7402-X) with the methods of report gene, Realtime PCR and Western blot. Meanwhile, the expression of Capn4 was detected after inhibition of HBx in HepG2-X (H7402-X) with siRNA.2. Transiently transfected with HBV X gene into HepG2 (H7402) cells and detected the expression of Capn4 with report gene, Realtime PCR and Western blot after 24h.3. With the model of the Human hepatoma cell line, HepG2, we have deteced the role of HBx and Capn4 in the migration of hepatoma cell through wound healing assay.4. Analyzed promoter of Capn4 using bioinformatics software and discovered that the promoter regions of Capn4 contain a sequence which matches the canonical Nuclear Factor-κB (NF-κB) binding site. Then, we used siRNA and specific inhibitor to silence and inhibit the activation of NF-κB in HepG2-X and H7402-X cells, and detect the expression of Capn4 through report gene assay and Western blot. Moreover, with single nucleotide mutation technique, the potential NF-κB combined sites were mutated in the Capn4 promoter sequence. The effect of HBx on mutant Capn4 promoter was detected by report gene.RESULTS 1. Capn4 expression in HepG2-X (H7402-X) is significantly higher than that in HepG2 (H7402), and it could be significantly depressed by the siRNA targeting HBV X gene.2. The expression levels of Capn4 in HepG2 (H7402) cells which transiently transfencted with HBV X gene for 24h were remarkably upregulated.3. The migration ability of HepG2-X, in which HBx stably expressed, is significantly higher than that of HepG2, and the siRNA targeting HBV X gene could significantly depress the migration ability of HepG2-X. Meanwhile, the migration ability of HepG2-X decreased significantly after Capn4 was silenced with siRNA.4. The expression of Capn4 can be obviously decreased by silence and inhibition of NF-κB activation in HepG2-X and H7402-X cells. Moreover, the HBx cannot activate mutated Capn4 promoter in HepG2 and H7420 cells.CONCLUSION In both of HepG2 and H7402 cell lines, HBx could upregulate the expression levels of Capn4 though the activation of NF-κB. The upregulation of Capn4 is involved in the HBx induced migration of HCC cells.