The present study suggests that compared with HBsAg, HBx and HBc proteins could more significantly inhibit MICA expression

The present study suggests that compared with HBsAg, HBx and HBc proteins could more significantly inhibit MICA expression. 0.01, compared with HepG2 cells (paired Rabbit Polyclonal to KNG1 (H chain, Cleaved-Lys380) 0.01; * 0.05, compared with HepG2-N1 (paired 0.01; * 0.05, compared with negative control (paired 0.01; * 0.05, compared with negative control (paired directly binding to the CpG island of MICA/B promoter Next, we attempted to investigate the role of HBc in the regulation of MICA/B. The HBc protein has been shown to directly bind to promoter regions containing CpG islands [9, 10]. Thus, we predicted two CpG islands in the MICA promoter by using the Emboss cpgplot database (Figure ?(Figure6A).6A). To determine whether Efonidipine hydrochloride monoethanolate the HBc protein can directly bind with CpG islands in the MICA promoter, chromatin fragments from HepG2.2.15 cells were immunoprecipitated with an anti-HBc antibody. DNA from the immunoprecipitation was isolated, and the two CpG regions were amplified. PCR analysis Efonidipine hydrochloride monoethanolate showed that the HBc protein could bind to CpG island 2 but not CpG island 1 (Figure ?(Figure6B).6B). In addition, we used the P1, P2 or P3 primer to amplify the MICA promoter with the same DNA from the immunoprecipitation assay, Efonidipine hydrochloride monoethanolate but the MICA promoter was not detected (Figure ?(Figure6C).6C). Furthermore, the GATA-2 or GATA-3 protein were not be detected from complexes immunoprecipitated with an anti-HBc antibody by immunoblot analysis in HepG2.2.15 cells (Figure ?(Figure6D).6D). The results indicated that the HBc protein could not bind to the GATA-2 or GATA-3 binding sites. Thus, the HBc protein inhibited MICA expression directly binding to the CpG island 2 of the MICA promoter. As it Efonidipine hydrochloride monoethanolate was shown in Figure S2, HBc also downregulated the expression of MICB, thus, by using the Emboss cpgplot database, we predicted a CpG island in the MICB promoter (Supplementary Figure S4A). ChIP analysis showed that the HBc protein could also bind to CpG island of MICB promoter (Supplementary Figure S4B). Open in a separate window Figure 6 HBV core protein inhibits MICA expression directly binding to the CpG island of MICA promoterA. CpG islands were predicted in the MICA promoter. B. and C. Soluble chromatin was immunoprecipitated with an anti-HBc antibody. PCR was used to amplify the MICA promoter containing CpG island isolated from the immunoprecipitated chromatin. D. Lysates from HepG2.2.15 cells were immunoprecipitated with an anti-HBc or control Ig, and then the sample was subjected to Western blotting with indicated antibodies. DISCUSSION The precise mechanism for HBV-induced down-regulation of NKG2D ligands on hepatoma cells remains unclear. In the present study, we found for the first time that HBV infection could promote the expression of transcription factors GATA-2 and GATA-3, which specifically suppressed MICA/B expression directly binding to the MICA/B promoter. Moreover, the HBx protein acted as a and contributed to the GATA-2 and GATA-3-mediated suppression of MICA expression. HBc protein could suppress MICA/B expression directly binding to the CpG islands of the MICA or MICB promoter (Figure ?(Figure77). Open in a separate window Figure 7 Working model for HBV suppression of MICA/B expression on hepatoma cellsChronic HBV infection up-regulates the expression of transcription factors GATA-2 and GATA-3 in HBV+ hepatoma cells. GATA-2 and GATA-3 directly target the MICA/B promoter to inhibit MICA/B transcription. Meanwhile, HBx binds with GATA-2 or GATA-3 and acts as a co-regulator contributing to the GATA-2 and GATA-3-mediated down-regulation of MICA expression. HBc directly binds to the the CpG island of the MICA or MICB promoter and inhibits MICA/B expression. NKG2D ligands are not expressed on most normal cells, but they are induced in tumor cells and virus-infected cells. Increasing evidence has shown that cellular stress, Efonidipine hydrochloride monoethanolate infection or tumorigenesis promote the expression of NKG2D ligands [21, 22]. The modulation process may occur at different stages, including transcription, RNA stabilization, protein stabilization and the cleavage.