Objective: To investigate the down-regulating effect of HBV pre-S2 protein upon insulin receptor (INSR) gene promoter.
Methods: Eukaryotic expression plasmid pcDNA3.1(-)-preS2 and report plasmid pCAT3-INSRp containing INSR gene promoter upstream of CAT gene were constructed with routine molecular biological methods and were confirmed by endonuclease digestion analysis and sequencing. Then HepG2 cells were transfected with pcDNA3.1(-)-preS2 plasmid and the total RNAs were examined with relative quantitative real-time PCR to confirm the expression of HBV pre-S2 protein. Later HepG2 and Huh7 cells were transfected with pCAT3-INSRp at various doses (0 - 2.0 microg). The choloraphenical acetyltransferase (CAT) activity of transfected cells was detected with CAT-ELISA to generate the dose-effect curve of pCAT3-INSRp. Lastly pCAT3-INSRp (1.0 microg) and pcDNA3.1(-)-preS2 (1.0, 1.5, 2.0, 2.5, 3.0 microg) were co-transfected into HepG2 and Huh7 cells respectively to study the regulation effect of pre-S2 protein upon INSR promoter. Meanwhile pre-S2 monoclonal antibody was added into additional cells transfected with 2.0 microg of pcDNA3.1(-)-preS2 plasmid to evaluate the effect when pre-S2 protein was blocked.
Results: The mRNA of pre-S2 protein could be detected with real-time PCR indicating that pre-S2 protein was properly expressed in pcDNA3.1(-)-preS2-transfected cells. The expression of CAT increased proportionally with the incremental doses of pCAT3-INSRp. It suggested that the INSR gene promoter had its transcription activity. After co-transfection of pCAT3-INSRp and pcDNA3.1(-)-preS2, the CAT expression in HepG2 cells, comparing with that of controls, were 69.8%, 60.1%, 19.7%, 10.3%, 5.6% (36 h) and 68.6%, 56.0%, 10.3%, 8.6%, 3.2% (72 h) respectively. When pre-S2 monoclonal antibody was added into the supernatant of HepG2 cells transfected with 2.0 microg of pcDNA3.1(-)-preS2, the CAT expression was partly restored to 55.4%(36 h)and 69.7%(72 h)of controls. The similar results were observed in Huh7 cells.
Conclusion: The pre-S2 protein down-regulates the activity of INSR gene promoter so as to reduce the expression of INSR. It may partly elucidate the pathogenesis of hepatogenous diabetes at the molecular level.