The Role Of HCV NS5A In Insulin Signal Pathway

BackgroundPersistent infection with hepatitis C virus (HCV), which is hepatotropic blood-borne chronic virus, is a major cause of chronic liver disease and leads to the development of cirrhosis and hepatocellular carcinoma. Data from WHO exhibited that approximately170million (about3%) people are chronically infected worldwide. In China,3.2%infections were recorded and increases in parallel with year, as does the number of progression towards hepatocellular carcinoma.Chronic HCV is treated with a glycoprotein commonly known as interferon (INF) alpha in combination with other auxiliary drugs and it is considered the backbone of therapy. However,an absolute therapy of hepatitis C remains problematic in a large majority of patients, and there is no effective preventative vaccine yet.Thus,HCV infection has become an important social problem and also been paid more and wide attention.But the mechanisms of HCV replication, persistence, and pathogenesis remain poorly understood.Hepatitis C virus is the only member of Hepacivirus of the Flaviviridae family. The genome of HCV encodes structural proteins and nonstructural protein, among which NS5A is an important non-structural proteins. NS5A is a multi-functional protein that is expressed in basically phosphorylated(p56) and in hyperphosphorylated (p58) forms and associates with a variety of signalling proteins in the host. Recent study found that the interaction between NS5A and Insulin/PI3K/Akt.Insulin signalling pathway (Insulin/PI3K/Akt) carries our its biological effects such as metabolism, cell cycle regulation, together with cell growth and apoptosis. This means that NS5A may play its role in the course of HCV infection by involving in insulin signalling pathway.Under normal circumstances, this signalling pathway is subject to strict regulation.As the homeostasis is broken,it may cause different diseases. Structural alteration of key factor PI3K(the regulatory subunit p85and the catalytic subunit p110), Akt and PTEN in Insulin/PI3K/Akt signalling pathway causes carcinoma encountered with disorder of metabolism, cell cycle, cell growth and apoptosis. HCV protein NS5A, has shown to activate Serine/threonine kinase(also known as Akt/protein kinase B) through integrating and triggering of PI3K, can break the homeostasis of signalling pathway so as to induce antivirus resistance and hepatocellular carcinoma. Major attention has been devoted to analyze the molecular interaction between HCV protein NS5A and the homeostasis of Insulin/PI3K/Akt signalling transduction and unravel the role of HCV in hepatocellular carcinoma.PTEN is another important tumor suppressor gene after p53, the reduction of whose gene function may lead to a variety of tumors. Previous studies reported that PTEN was less expressed in HCC. However,PTEN, a main negative regulator of insulin signaling,can inhibit Insulin/PI3K/Akt signalling in contrast to the role of HCV in Insulin/PI3K/Akt signalling pathway. This article researches on the role of PTEN on the homeostasis of insulin signalling pathway and the damage of HCV protein NS5A to the homeostasis of insulin signaling, indicating that PTEN and NS5A can change the homeostasis of insulin signalling and then lead to cell survival or apoptosis from positive and negative aspects respectively.Part one:The role of PTEN on the homeostasis of Insulin/PI3K/Akt pathwayObjectiveThe function of PTEN is weaken in HCC.Therefore,we study the role of PTEN on the homeostasis of Insulin/PI3K/Akt pathway firstly.We use the human hepatoma cell line HepG2of stable metabolic state as research models, stimulate them with insulin, increase or silence the expression of the upstream negative regulator gene PTEN of Insulin/PI3K/Akt pathway, and reversely study the homeostasis of Insulin/PI3K/Akt signalling pathway from the protein level with Western blot technology.Methods1.Cultivate HepG2cell line in vitro routinely, and then passage to12well plates evenly.When the cell grow to approximately90%, do serum starvation for24hours and then cell lysis.10min before cells lysis, we divided the cells into insulin stimulation group or the control group, according to adding insulin to stimulate (insulin concentration is2ul/ml, diluted in serum free DMEM medium) or adding the same volume of serum-free DMEM. Do cell lysis, extract total protein and measure the p-Akt protein expression with Western blot.2. Cultivate HepG2cell line in vitro routinely, and divide the cells randomly into two groups of PTEN wt Group and PTENC124S group, transfected with the human wild-type PTEN eukaryotic expression plasmid (pSvEGFP-PTEN) or phosphatase domain mutant plasmid pSvEGFP-C124S. Do transfection and serum starvation as above. Do cell lysis48hours after transfection,stimulate cells with insulin for ten minutes and then extract the total protein. Detect the p-Akt protein levels by Western blot analysis. 3. Divide the cultivated HepG2cell line randomly into two groups, the PTEN siRNA group and the control group, transfected with PTEN siRNA and its control siRNA.transfection, serum starvation as above. Do transfection and serum starvation as above. Do cell lysis48hours after transfection,stimulate cells with insulin for ten minutes and then extract the total protein. Measure the two proteins of p-Akt and PTEN expression with Western blot.4. Statistical Analysis. Data were expressed as the geometric means±standard error of mean of experiments. Difference between two groups was analyzed by t test or Wilcoxon rank sum test. P<0.05was considered to be significant.Results1. We found that the p-Akt protein expression quantity of insulin stimulation group was more than the control group (P<0.010, the two groups of gray-scale ratio were0.221±0.136and0.000±0.000). Western blot with β-actin as internal reference.2. Western blot results showed that PTENC124S group increased the level of Akt phosphorylation compared with PTEN wt. Group (gray odds ratios were0.360±0.141and0.084±0.055, P<0.05).3. The results of Western blot indicated that the PTEN protein expression of PTEN siRNA group is lower than the control group (P<0.002), the corresponding p-Akt expression quantity is significantly higher (P<0.02).Conclusion1.Insulin can induce Insulin/PI3K/Akt signalling pathway activation.2.The negative regulatory factor of Insulin/PI3K/Akt signalling pathways, PTEN point mutation or knockdown, can cause the activation of downstream signalling molecules Akt.3.Under normal circumstances,Insulin/PI3K/Akt signal pathway is tightly regulated, exogenous intervention to make any link of the signalling pathway blocked or out of control, the steady-state will be broken into excessive inhibition or excessive activation.Part two:The role of HCV NS5A in Insulin signal pathwayObjectiveThe part above indicates the role of PTEN on the homeostasis of Insulin/PI3K/Akt pathway.We transfected HepG2cells with the hepatitis C virus NS5A plasmid, to explore the role of NS5A in regulation of the PI3K/Akt pathway, and provide an implication for clinical events such as hepatitis C chronic persistent,HCC and antiviral resistance.Methods1. Divide the cultivated HepG2cell line randomly into two groups.One group was transfected with NS5A plasmid, and the other was transfected with its control empty vector. Do serum starvation for24hours and then cell lysis.10min After sitimulated with insulin,cells were lysed conventionally and total protein was extracted48hours after transfection. Measure the p-Akt protein expression with Western blot at last.2. Divide the cultivated HepG2cell line randomly into two groups, namely the NS5A group and control group, transfected with NS5A plasmid or control empty vector.48hours after transfection,extract the total protein. Adopt immunoprecipitation assay:immunoprecipitate firstly with total HepG2cells protein of NS5A group or a control group and PI3K subunit p85polyclonal antibody and then do Western blot with anti-pTyr antibody to compare p85tyrosine phosphorylation level between the two groups.3. Divide the cultivated HepG2cell line randomly into two groups, namely the NS5A group and control group, transfected with NS5A plasmid or control empty vector.48hours after transfection extract the total protein. Adopt immunoprecipitation assay:immunoprecipitate firstly with total HepG2cells protein of NS5A group or a control group and PI3K catalytic subunit p85polyclonal antibody, and then do Western blot with anti-pTyr antibody to compare the binding ability of the catalytic subunit p110and regulatory subunit p85of PI3K between the two groups.4. Statistical Analysis. Data were expressed as the geometric means±standard error of mean of experiments. Difference between two groups was analyzed by t test or Wilcoxon rank sum test. P<0.05was considered to be significant.Results1. Western blot results showed that the p-Akt protein level of the NS5A group was significantly up-regulated, compared with the control group.(gray scale ratio were0.811±0.131and0.205±0.066, P<0.002).2. The result of immunoprecipitation indicated that the group transfected with NS5A plasmid had a higher p85tyrosine phosphorylation levels than the control group (gray scale ratio of1.055±0.189and0.479±0.147,0.014).3. The result of co-immunoprecipitation assay showed that Binding effects of the p85and p110protein had no significant differences in quantity between the NS5A group and the control group (gray scale ratio of0.543±0.286and0.654±0.195, respectively, P>0.05).ConclusionThe HCV protein NS5A can bind with the PI3K regulatory subunit p85protein and then activate Insulin/PI3K/Akt signalling pathway. However the combination of HCV protein NS5A and the p85protein did not affect the combination of PI3K regulating subunit p85and the catalytic subunit p110. So we hypothesized that HCV protein NS5A regulating the PI3K signalling pathway may more than via PI3K/Akt pathway.So we draw forth another machenism of HCV regulating the PI3K signalling pathway involving PTEN.