The Effect Of HT-1 Protein Tax On Autophagy Induced By Extracellular HMGB1

BackgroundAutophagy,a process of cell survival,mainly degrade damaged organelles and long-lived proteins as a lysosomal degradation pathway and play a very important role for cellular homeostasis.HMGB1 is a classic damage associated molecular patterns(DAMP),which can regulate cellular growth and differentiation,inflammation and metabolism.Our laboratory studies have previously showed that extracellular HMGB1 can promote autophagy in HTLV-1 infected T cells.Tumor protein Tax encoded by HTLV-1 virus controls many important cell signaling pathways,also has the role in regulating of autophagy.However,it is unclear that the effect of Tax on autophagy induced by extracellular HMGB1.PurposeTo study the role of Tax protein on autophagy;To study the effect of Tax on autophagy induced by extracellular HMGB1;And to explore the possible mechanism.Methods1.Cell culture: HeLa cells were cultured in the incubator containing 5% CO2 and moderate humidity at 37 ? and used the culture medium containing 10% fetal bovine serum,2mML--glutamine,100 UG/mL streptomycin,and 100u/mL penicillin RPMI 1640 or DMEM medium.Cell status and density were observed every day,and the fresh medium was replaced 1 time every 2 days,and the cells were passaged for 1 time in 3 days;2.Plasmid extraction: Coat the LC3B-EGFP-mCherry fluorescent plasmid bacteria liquid,pCMV-Neo,pCMV-Tax,pCMV-Tax-m47,pCMV-Tax-m22 plasmid bacteria liquid on the sheet,pick the positive monoclonal,shake the bacteria in the bottlesand and preservate the fungi,and then extract plasmids according to the descriptions of Plasmid.Extraction Kit of endotoxin free from TIAN GEN,finally check the quality and purity ofextracted plasmids.3.Cells transfection: LC3B-EGFP-mCherry,pCMV-Neo,pCMV-Tax,pCMVTax-m47 and pCMV-Tax-m22 plasmids were transfected into Hela cells with lipofectamine2000 reageny.After the transfection for 6 hours,the medium was replaced with preheating complete medium which contains serum but not contains double antibody.After 48 hours,collected the cells and extracted proteins to detect the expressions of LC3 and p62 by Western blot or to observe numbers of the formed autophagosome by confocal microscope.4.Drug treatment: Hela cells were stimulated using the final concentration of 1 g/mL rhHMGB1 and used Western blot and confocal microscopy to observe the changes of the relevant indicators.5.Detection of indicators: Proteins were extracted from rhHMGB1 treatment group and the control group of HeLa cells and analyze the expressions of LC3 and p62 proteins by Western blot;After transfection into plasmids and treated with rhHMGB1,Hela cells were observed for autophagosome formation.using confocal microscopy.6.Statistical analysis: SPSS 19.0 software was used to process the data.Results were showed with the mean�standard deviation(? x�s),the t-test was used for comparison of two groups,P < 0.05 was thinked for a statistically significant difference.Results1.Dual luciferase report gene detection results showed that the expression product NF-kappa-B activity of the eukaryotic expression plasmid pCMV-Tax and pCMV-Tax-m47 was significantly higher than that of pCMV-Neo and pCMV-Tax-m22 plasmid.2.Western Blot results showed that after transfection into pCMV-Tax or pCMV-Tax-m47 plasmid in Hela cells,compared with the control group,the ratio of LC3-II /LC3-I was significantly increased.And the ratio of LC3-II /LC3-I was not significantly changed after the transfection of pCMV-Tax-m22 plasmid.3.Western blot results showed that after Hela cells were treated with rhHMGB1 and plasmid pCMV-Tax or pCMV-Tax-m47 transfection,compared with treated withrhHMGB1 only,the ratio of LC3 II /LC3-I was more obvious increased.4.Confocal results showed that after rhHMGB1 treatment or transfection into the pCMV-Tax and pCMV-Tax-m47 plasmids,numbers of autophogome were increased significantly and some of them were gathered into clusters.When the two factors coexisted,numbers of autophogome were more.5.Confocal results showed that the eukaryotic expression plasmid LC3B-EGFP-mCherry and pCMV-Tax were transfected into Hela cells using rhHMGB1 treatment,compared with the control group,experimental group could see the yellow autophagosomes only and control group can see yellow and red autophagosomes.ConclusionExpression of HTLV-1 protein tax can promote autophagy induced by extracellular HMGB1.This effect may be realized through NF-kappa-B signal pathway.