Ji Dong, Han Ping, Zhang Jian, Shao Qing, Liu Yan, Wang Lin, Chen Guo-feng
Military Postgraduate Medical School, Department of Infectious Diseases, Beijing Hospital of PLA, China.
Zhonghua Yi Xue Za Zhi. 2009 Nov 24;89(43):3069-73.
To investigate the down-regulating effect of HBV pre-S2 protein upon insulin receptor (INSR) gene promoter.
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.
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.
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.
研究乙型肝炎病毒（HBV）前S2蛋白对胰岛素受体（INSR）基因启动子的下调作用。
采用常规分子生物学方法构建真核表达质粒pcDNA3.1(-)-preS2及含INSR基因启动子上游序列并位于氯霉素乙酰转移酶（CAT）基因上游的报告质粒pCAT3-INSRp，经酶切分析及测序鉴定。将pcDNA3.1(-)-preS2质粒转染HepG2细胞，采用相对定量实时荧光定量PCR检测总RNA，以确认HBV前S2蛋白的表达。随后，将不同剂量（0～2.0 μg）的pCAT3-INSRp转染HepG2和Huh7细胞。采用CAT-ELISA法检测转染细胞的氯霉素乙酰转移酶（CAT）活性，绘制pCAT3-INSRp的剂量效应曲线。最后，分别将pCAT3-INSRp（1.0 μg）与pcDNA3.1(-)-preS2（1.0、1.5、2.0、2.5、3.0 μg）共转染HepG2和Huh7细胞，研究前S2蛋白对INSR启动子的调控作用。同时，在转染2.0 μg pcDNA3.1(-)-preS2质粒的细胞中加入前S2单克隆抗体，评估前S2蛋白被阻断时的效应。
实时荧光定量PCR可检测到前S2蛋白的mRNA，表明前S2蛋白在pcDNA3.1(-)-preS2转染细胞中正确表达。CAT表达随pCAT3-INSRp剂量增加而成比例增加。提示INSR基因启动子具有转录活性。pCAT3-INSRp与pcDNA3.1(-)-preS2共转染后，HepG2细胞中CAT表达与对照组相比，36 h时分别为69.8%、60.1%、19.7%、10.3%、5.6%，72 h时分别为68.6%、56.0%、10.3%、8.6%、3.2%。在转染2.0 μg pcDNA3.1(-)-preS2的HepG2细胞上清中加入前S2单克隆抗体后，CAT表达部分恢复至对照组的55.4%（36 h）和69.7%（72 h）。在Huh7细胞中观察到类似结果。
前S2蛋白下调INSR基因启动子活性，从而降低INSR的表达。这可能在分子水平上部分阐明了肝源性糖尿病的发病机制。
