Studies On Mechanisms Of PTEN In Regulation Of HCV Associated Insulin Resistance And In Inhibition Of DENV Infection

PTEN(phosphatase and tensin homolog deleted in chromosome 10, PTEN), also known as MMACl, TGF-B or TEPl, was discovered in 1997, which was a tumor suppressor genelocated in chromosome 10(10q23.3), and so far also it was the only one that has both protein phosphatase activity and lipid phosphatase activity in the body. In a variety of tumors, such as prostate cancer, breast cancer and sporadic glioblastoma, the deleted mutation rate of PTEN is high.PTEN protein consists of 403 amino acid residues,which N- terminal contains phosphatase domain and C- terminus has a PDZ domain protein binding sequence; PTEN as a protein phosphatase, can degrade inositol triphosphate, and therefore PTEN is a very effective negative regulator of P13K-Akt pathway, which may affect insulin signaling in the target tissues. Meanwhile, PTEN can regulate the mammalian target of rapamycin(mammalian target of rapamycin, m TOR)activity and process of autophagy. In the study, the role of PTEN in hepatitis C virus(hepatitis C virus, HCV) associated insulin resistance and its regulation of autophagy activity against dengue virus(dengue virus, DENV) infection were explored, to provide basis for the understanding of pathogenesis and prevention measures of HCV and DENV.(A) The role of PTEN in HCV-related insulin resistance and its of insulin and its mechanisms studyHCV is a hepatotropic virus spread primarily through blood, according to World Health Organization statistics, approximately 170 million people were infected with HCV,and the infection rate was up to 3%. In our courtry, the anti-HCV positive rate in healthy people is 0.7-3.1%, which isabout 38 million people, and the infection rate is still an increasing trend. HCV genome has high variability and variety of host immune evasion mechanisms to makeit not effective to remove the virus, resulting in about 50-80% of infected people to develop into chronic hepatitis C, 20-30% of which will develop into cirrhosis. In patients with cirrhosis, 1-4% of which evolved to HCC(hepatacellular carcinoma, HCC) annually. Although pegylated interferon and ribavirin, plus the latest triple direct antiviral therapy for HCV infection can greatly improve sustained virologic responses, but it still faces wih high cost of drug, long period of treatment, side effects and so on, also there is no effective vaccine available.Chronic HCV infection can lead to chronic liver inflammation, necrosis and fibrosis,some patients may develop to cirrhosis or liver cancer. Studies have shown that 25%-50%patients with chronic hepatitis C have abnormal levels of glucose and lipid metabolism.The risk of HCV infection developing into insulin resistance(insulin resistance, IR) and type 2 diabetes(type 2 diabetes mellitus, T2DM) is getting higher and higher. Chronic HCV infection is closely related with IR, leading to a vicious cycle, so that these patients become refractory hepatitis C patients in clinical practice. Meanwhile, IR is the central link of hepatic steatosis, cirrhosis and the development of hepatocellular carcinoma, which is also an independent predictor of HCC process.PTEN is a specific tyrosine phosphatase protein, which catalyses the degradation of inositol triphosphate PIP3, and PIP3 is the product of phosphatidylinositol kinase PI3 K,which may activate the downstream Akt to pass insulin signaling, and is an important factor in insulin signal pathway, promoting the function of glucose transporter-4(glucose transporter-4, GLUT-4). In 3T3-L1 adipocytes, the role of endogenous PTEN is to weaken the role of insulin on glucose, causing insulin resistance in adipocytes. In skeletal muscle cells, it also found that PTEN can block insulin signaling pathway, resulting in insulin resistance. Studies found that PTEN function decreases in HCV caused HCC, while PTEN can be involved in insulin resistance by decreasing PI3 K / Akt pathway.In the study, we found that HCV infection can promote insulin receptor substrate-1(insulin receptor substrate-1, IRS-1) 307 phosphorylation of Ser at the position, and thus negatively inhibiting insulin signaling pathway IRS-1/PI3K/Akt, to promote IRS-1degradation. Further studies showed that HCV infection may reduce sensitivity to exogenous insulin stimulation. Huh7.5.1 hepatoma cells were stimulated with insulin, and in HCV infection group Akt 473 and Ser at position Ser at position GSK3 β 9phosphorylation levels were significantly reduced. To explore the role of PTEN, as an important negative regulator of PI3K/Akt signaling pathway in the occurrence of HCV-related insulin resistance, we used different multiplicity of infection(MOI = 1, 2, 3)of HCVcc to infect Huh7.5.1 cells for 48 h, and the results suggested that HCV infection downregulated significantly PTEN m RNA and protein expression levels. We further found that PTEN overexpression decreased IRS-1 Ser 307 phosphorylation leveland rescued IRS-1 degradation. In PTEN knockdown group p IRS-1Ser307 expression levels were significantly increased, while the level of IRS-1 significantly reduced. These results suggest that upregulated p IRS-1Ser307 and downregulated IRS-1 caused by HCV 2a infection may be in a PTEN-dependent manner. Then, we constructed p JFH1-core plasmidto transfecte Huh7.5.1 cells and found that HCV core protein can reduceevels of PTEN,while increase p IRS-1Ser307 to promote IRS-1 degradation. The results suggest that, HCV2 a core itself can reduce PTEN protein expression levels, while raising p IRS-1Ser307 expression to promote of IRS-1 degradation, the key molecules in insulin signaling, and thus participate in insulin resistance.We have studied the relationship between PTEN and cellular insulin signaling pathway, to further explore the role of PTEN in the occurrence of HCV-related insulin resistance, and the impact of HCV core protein on PTEN and insulin signaling pathways,which provides new ideas and therapeutic targets for the study of HCV and insulin resistance.(B) PTEN activity through inhibition of autophagy down dengue virus infectionDengue virus(dengue virus, DENV) is a single positive-strand RNA virus belonging to flavivirus family. Dengue virus infection often causes a self-limiting dengue fever(dengue fever, DF), and severe patients may develop to dengue hemorrhagic fever(dengue hemorrhagic fever, DHF) and dengue shock syndrome(dengue shock syndrome, DSS). In recent years, with the increase in global warming and population movements, dengue virus infection has an outbreak of trends, and has become a serious public health problem. So far,there is no specific treatment for drugs and effective vaccines available. Therefore,pathogenesis and prevention of dengue virus infection problems are still to be solved.Autophagy is the process of intracellular lysosomal to degrade its component in eukaryotic cells and plays an important role in the intracellular homeostasis. More and more studies show that autophagy is closely related with pathogens. Some viruses inhibit autophagy by its encoded proteinsto escape autophagy scavenging; while some single positive-strand RNA viruses, including dengue flavivirus genus, to utilize autophagic vacuoles as a replication site and promote their proliferation by inducing autophagy.PTEN, as an important negative regulator of m TOR signaling pathway molecules, is involved in the regulation of autophagy. To investigate PTEN, autophagy and dengue virus type 2 infection and theie possible mechanisms, human umbilical vein endothelial cells(human umbilical vein endothelial cells, HUVEC) were used as DENV infected target cells in our study. In order to explore the dengue virus infection effect on cellular autophagy, weuse different M.O.I of DENV to infect HUVEC cells for 48 h, and Western-blot was used to detect LC3 II / LC3 I ratio changes, for that microtubule-associated protein light chain 3(microtubule-associated protein light chain 3, LC3) is the specific autophagy marker. The results show that with the increase of infection.O.I, the ratio of LC3 II / LC3 I increases,suggesting that DENV infection can induce autophagy in HUVEC cells and autophagic activity positively correlates with the amount of virus infection. In order to study the role of PTEN, as autophagy important regulatory protein, in DENV infection, we use different M.O.I of DENV to infect HUVEC cells for 48 h, and Western blot and real-time PCR detection were used to detec PTEN protein expression and m RNA levels, respectively. The results show that with the increase of M.O.I, the level of PTEN is significantly reduced.To further investigate the effects of PTEN on dengue virus infection, we constructed p Lenti-PTEN and p Lenti-sh PTEN plasmid and packaged lentiviruses to infect HUVEC cells for the purpose of overexpression or knockdown of PTEN expression levels, and after DENV infection, western-blot and quantitative PCR were used to detect intracellular DENV capsid protein and m RNA levels. Then indirect immunofluorescence was used to detect DENV progeny infectivity in the cell culture supernatant. The results suggest that over-expression of PTEN can significantly increase the ratio LC3 II / LC3 I and promote specific degradation of P62 protein, which further promote the replication of DENV capsid and release of progeny virus particles. While the silencing of PTEN inhibits autophagy activity, namely LC3 II / LC3 I ratio decreased with increased P62 protein levels, so that the replication level of DENV and the content of progeny virus particles in the supernatant are significantly reduced.The results of our study suggest that PTEN down-regulation induced by DENV infection in HUVEC cells may be a host cell protective responses itself against viral infection, which establish theoretical basis a for further research on the interaction between dengue virus and the host cellas well as providing new ideas on prevetional strategies of DENV infection.