PTEN基因沉默对小鼠原代肝细胞Akt/GSK-3β信号通路的影响
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摘要
目的探讨第10号染色体缺失性磷酸酶-张力蛋白同源物蛋白(PTEN)对小鼠原代肝细胞蛋白激酶B/糖原合酶激酶-3β(Akt/GSK-3β)信号通路的影响。方法采用两步胶原酶灌注法分离小鼠原代肝细胞,均分为4组:阴性对照组(NC-C组)、阴性对照白细胞介素-6(IL-6)组(NC-IL组)、siR-996对照组(996-C组)、siR-996与IL-6组(996-IL组)。采用HiPerFect转染试剂将siRNA-996转入小鼠原代肝细胞,24h后提取蛋白,通过蛋白质印迹法(Western blot)检测各组Akt、磷酸化Akt(p-Akt)、GSK-3β、磷酸化GSK-3β(pGSK-3β)、PTEN蛋白水平。结果在PTEN蛋白水平上,与NC-C组相比,NC-IL组升高(P<0.05),996-C组和996-IL组降低(P<0.01)。在p-Akt/Akt上,与NC-C组相比,NC-IL组降低(P<0.05),996-C组和996-IL组升高(P<0.05)。在p-GSK-3β/GSK-3β上,与NC-C组相比,NC-IL组降低(P<0.05),996-C组和996-IL组升高(P<0.01)。结论 PTEN基因沉默可逆转IL-6对小鼠原代肝细胞Akt/GSK-3β信号通路的抑制,增强小鼠原代肝细胞Akt/GSK-3β信号通路的表达。
Objective To investigate the effect of phosphatase and tensin homologue deleted on chromosome 10(PTEN)on protein kinase B/glycogen synthase kinase-3β(Akt/GSK-3β)signaling pathway in mouse primary hepatocytes.Methods The mouse primary hepatocytes were isolated by two-step collagenase perfusion and divided into four groups on average:the negative control group(the NC-C group),the negative control IL-6 group(the NC-IL group),the siR-996 control group(the 996-C group)and the siR-996 and IL-6 group(the 996-IL group).siRNA-996 was transfected into mouse primary hepatocytes by HiPerFect transfection reagent.After 24 hculture,the protein was extracted,and the protein levels of Akt,p-Akt,GSK-3β,pGSK-3βand PTEN in each group were detected by Western blot.Results Compared with the NC-C group,the level of PTEN protein increased in the NC-IL group(P<0.05),while decreased significantly in the 996-C group and the 996-IL group(P<0.01).Compared with the NC-C group,p-Akt/Akt was decreased in the NCIL group(P<0.05),while it was increased in the 996-C group and the 996-IL group(P<0.05).Compared with the NC-C group,p-GSK-3β/GSK-3βwas decreased in the NC-IL group(P<0.05),while it was increased significantly in the 996-C group and the 996-IL group(P<0.01).Conclusion PTEN gene silencing can reverse the inhibition of IL-6 on Akt/GSK-3βsignaling pathway in mouse primary hepatocytes,and enhance the expression of Akt/GSK-3βsignaling pathway in mouse primary hepatocytes.
Objective To investigate the effect of phosphatase and tensin homologue deleted on chromosome 10(PTEN)on protein kinase B/glycogen synthase kinase-3β(Akt/GSK-3β)signaling pathway in mouse primary hepatocytes.Methods The mouse primary hepatocytes were isolated by two-step collagenase perfusion and divided into four groups on average:the negative control group(the NC-C group),the negative control IL-6 group(the NC-IL group),the siR-996 control group(the 996-C group)and the siR-996 and IL-6 group(the 996-IL group).siRNA-996 was transfected into mouse primary hepatocytes by HiPerFect transfection reagent.After 24 hculture,the protein was extracted,and the protein levels of Akt,p-Akt,GSK-3β,pGSK-3βand PTEN in each group were detected by Western blot.Results Compared with the NC-C group,the level of PTEN protein increased in the NC-IL group(P<0.05),while decreased significantly in the 996-C group and the 996-IL group(P<0.01).Compared with the NC-C group,p-Akt/Akt was decreased in the NCIL group(P<0.05),while it was increased in the 996-C group and the 996-IL group(P<0.05).Compared with the NC-C group,p-GSK-3β/GSK-3βwas decreased in the NC-IL group(P<0.05),while it was increased significantly in the 996-C group and the 996-IL group(P<0.01).Conclusion PTEN gene silencing can reverse the inhibition of IL-6 on Akt/GSK-3βsignaling pathway in mouse primary hepatocytes,and enhance the expression of Akt/GSK-3βsignaling pathway in mouse primary hepatocytes.
引文
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[11]GIRAUD J,LESHAN R,LEE Y H,et al.Nutrient-dependent and insulin-stimulated phosphorylation of insulin receptor substrate-1 on serine 302correlates with increased insulin signaling[J].J Biol Chem,2004,279(5):3447-3454.
[12]CHU E C,TARNAWSKI A S.PTEN regulatory functions in tumor suppression and cell biology[J].Med Sci Monit,2004,10(10):RA235-241.
[13]LEE Y R,CHEN M,PANDOLFI P P.The functions and regulation of the PTEN tumour suppressor:new modes and prospects[J].Nat Rev Mol Cell Biol,2018,19(9):547-562.
[14]韩杨,程克棣,朱平.抑癌基因PTEN的研究进展[J].国外医学(药学分册),2006,33(4):241-245.
[15]HADDADI N,LIN Y,TRAVIS G,et al.PTEN/PTENP1:′Regulating the regulator of RTK-dependent PI3K/Akt signalling′,new targets for cancer therapy[J].Mol Cancer,2018,17(1):37.
[16]LESLIE N R,BENNETT D,LINDSAY Y E,et al.Redox regulation of PI3-kinase signalling via inactivation of PTEN[J].EMBO J,2003,22(20):5501-5510.
[2]VAN DEN BERGHE G,WILMER A,HERMANS G,et al.Intensive insulin therapy in the medical ICU[J].N Engl J Med,2006,354(5):449-461.
[3]YASUDA Y,FUKUSHIMA Y,KANEKI M,et al.Anesthesia with propofol induces insulin resistance systemically in skeletal and cardiac muscles and liver of rats[J].Biochem Biophys Res Commun,2013,431(1):81-85.
[4]ZHOU L,WANG L,YANG B,et al.Protective effect of pretreatment with propofol against tumor necrosis factor--induced hepatic insulin resistance[J].Exp Ther Med,2015,10(1):289-294.
[5]DRIESSEN G J,IJSPEERT H,WENTINK M,et al.Increased PI3K/Akt activity and deregulated humoral immune response in human PTEN deficiency[J].J Allergy Clin Immunol,2016,138(6):1744-1747.
[6]YUE S,LI J,LEE S Y,et al.Cholesteryl ester accumulation induced by PTEN loss and PI3K/AKT activation underlies human prostate cancer aggressiveness[J].Cell Metab,2014,19(3):393-406.
[7]BLEAU A M,HAMBARDZUMYAN D,OZAWA T,et al.PTEN/PI3K/Akt pathway regulates the side population phenotype and ABCG2activity in glioma tumor stemlike cells[J].Cell Stem Cell,2009,4(3):226-235.
[8]SEGLEN P O.Preparation of isolated rat liver cells[J].Methods Cell Biol,1976,13:29-83.
[9]CASCIANO D A.Development and utilization of primary hepatocyte culture systems to evaluate metabolism,DNAbinding,and DNA repair of xenobiotics[J].Drug Metab Rev,2000,32(1):1-13.
[10]DOU L,WANG S,SUI X,et al.MiR-301amediates the effect of IL-6on the AKT/GSK pathway and hepatic glycogenesis by regulating PTEN expression[J].Cell Physiol Biochem,2015,35(4):1413-1424.
[11]GIRAUD J,LESHAN R,LEE Y H,et al.Nutrient-dependent and insulin-stimulated phosphorylation of insulin receptor substrate-1 on serine 302correlates with increased insulin signaling[J].J Biol Chem,2004,279(5):3447-3454.
[12]CHU E C,TARNAWSKI A S.PTEN regulatory functions in tumor suppression and cell biology[J].Med Sci Monit,2004,10(10):RA235-241.
[13]LEE Y R,CHEN M,PANDOLFI P P.The functions and regulation of the PTEN tumour suppressor:new modes and prospects[J].Nat Rev Mol Cell Biol,2018,19(9):547-562.
[14]韩杨,程克棣,朱平.抑癌基因PTEN的研究进展[J].国外医学(药学分册),2006,33(4):241-245.
[15]HADDADI N,LIN Y,TRAVIS G,et al.PTEN/PTENP1:′Regulating the regulator of RTK-dependent PI3K/Akt signalling′,new targets for cancer therapy[J].Mol Cancer,2018,17(1):37.
[16]LESLIE N R,BENNETT D,LINDSAY Y E,et al.Redox regulation of PI3-kinase signalling via inactivation of PTEN[J].EMBO J,2003,22(20):5501-5510.