Effect Of HIV-1 Nef On Autophagy And Apoptosis Of U87 And SH-SY5Y Cells In HAD And Non-HAD Patients

OBJECTIVEHIV-1 is the pathogen of acquired immunodeficiency syndrome(AIDS),which can invade the central nervous system(CNS)and cause a series of nervous system diseases,the most serious of which is HIV associated dementia(HAD).At present,the specific pathogenesis of HAD is not clear,but many studies have shown that the occurrence and development of HAD are related to autophagy and apoptosis.LC3 exists in all stages of autophagy and is the most commonly used autophagic marker,in general,we study the transformation of LC3B-Ⅰ/Ⅱ and combine it with p62 protein to judge the autophagy state.Aspartate specific cysteine protease plays a key role in the process of apoptosis,caspase-9 and caspase-3 are considered to be the key enzymes of apoptosis.As a retrovirus,HIV-1 has high variability,and the variation of central and peripheral environment are different,so the variation of HIV-1 is probably related to the pathogenesis of HAD.The early negative factor(Nef)is the regulatory protein of HIV-1,it can down regulate the surface molecules of host cell membrane,down regulation of MHC-Ⅰ and MHC-Ⅱ,enhance virus replication and infectivity,promote the development of brain injury related to HIV-1 infection.At present,there are many studies on the cytotoxic effects of HIV-1 Nef protein on CNS,but the mechanism has not been fully explained.Therefore,it is of great significance to study the mechanism of CNS injury caused by Nef protein and the pathogenesis of HAD.In this study,HIV-1 nef gene from CNS source of temporal cortex(TC)and peripheral spleen(SPL)of HAD patient and non-HAD patient.The amino acid sites of Nef protein from different sources were analyzed.To study the effect of Nef protein from central and peripheral sites of HAD patient and non-HAD patient on the activity of human neuroastroglioma cell line(U87)and human neuroblastoma cells(SH-SY5Y).To further study its effect on autophagy and apoptosis,so as to provide scientific basis for the study of the pathogenesis of HAD.METHODS1.HIV-1 nef gene cloning and amino acid sequence analysis The HIV-1 nef gene was amplified by PCR using TC and SPL genomic DNA of a HAD and non-HAD patient as template.PCR products were analyzed by 1%agarose gel electrophoresis and connected to the clone plasmid pMD-19T.The conjugates were transformed into E.coli DH-5α and the positive clones were screened by ampicillin.After PCR identification of bacterial solution,sequencing was carried out.The sequences were compared and analyzed by BLAST,and the variation of amino acid sites was analyzed by Bioedit,DNA man and other biological analysis software.2.Construction of eukaryotic expression vector of pEGFP-N1-nefpMD-19t-nef recombinant plasmid containing nef gene from H-TC,N-TC,H-SPL and N-SPL and eukaryotic expression plasmid pEGFP-N1 were used BamH I and ECOR I to cleave and connect.After agarose gel electrophoresis,the target gene and pEGFP-N1 plasmid were recovered by agarose gel electrophoresis.After T4 ligase was linked,it was transformed into Escherichia coli DH-5α.PCR identification of bacterial solution by kanamycin screening positive clone.The plasmid was extracted and identified by enzyme digestion and DNA sequencing.3.Expression of HIV-1 Nef protein in U87 and SH-SY5 Y cells and its effect on cell activityThe recombinant eukaryotic expression plasmid pEGFP-N1-nef of Nef from H-TC,N-TC,H-SPL and N-SPL was transfected into U87 and SH-SY5Y cells,pEGFP-N1 empty carrier group was negative control,the normal cell group was blank control,and the expression of HIV-1 Nef protein was evaluated by green fluorescence.Western-blot was used to identify the expression of Nef protein,meanwhile,CCK-8 kit was used to detect the effect of Nef protein on the two cell activities.4.Effect of HIV-1 Nef protein on autophagy of U87 and SH-SY5Y cellsThe control group and U87,SH-SY5Y cells were obtained after 48h of transfection,western blot was used to detect the expression of LC3-Ⅱ and p62.The gray value of the target protein was quantitatively analyzed by Image J software,calculated the ratios of LC3-Ⅱ and p62/β-actin.Analyzed the effects of Nef protein from different sources on the expression of LC3-Ⅱand p62 in U87 and SH-SY5Y cells were.5.Effect of HIV-1 Nef protein on apoptosis of U87 and SH-SY5Y cells(1)Apoptosis observed by DAPI stainingThe cells transfected with pEGFP-N1-nef were fixed for 48h,then stained with DAPI staining solution and observed apoptotic bodies under fluorescence microscope.Each sample is provided with 3 parallel holes,pEGFP-N1 empty carrier group was negative control,the normal cell group was blank control.According to the morphological changes of the nucleus,cell apoptosis was judged.(2)Detection of caspase-9 and caspase-3The control group and U87,SH-SY5Y cells were harvested,Western blot was used to detect caspase-9 and caspase-3,the gray value of the target protein was quantitatively analyzed by Image J software,caspase-9 and 3/β-actin ratio were calculated.The effects of Nef protein from different sources on the expression of caspase-9 and caspase-3 in U87 and SH-SY5Y cells were analyzed.6.Statistical analysisSPSS 17.0 software was used to deal with the experimental results,the experimental data are described by mean±standard deviation(x±s),and statistical tested by ANOVA(α=0.05).RESULTS1.HIV-1 nef gene cloning and amino acid sequence analysisHIV-1 nef gene was successfully cloned from TC and SPL of HAD and non-HAD patients.The sequence of HIV-1 nef gene was confirmed by blast analysis,two HAD and non-HAD patients were confirmed to be infected with HIV-1B subtype.Compared with the standard sequence HXB2,the results of amino acid site analysis showed that there were 35 mutations in TC and SPL in HAD patients.There were 36 mutations in TC and 34 in SPL in non-HAD patients.In addition,the mutations sites of Nef amino acids in CNS and peripheral tissues of HAD patient and non-HAD patient were different.Some of the mutations sites were found in CNS and peripheral tissues,some only in CNS,some only in peripheral tissues.2.Construction of eukaryotic expression vector of pEGFP-N1-nefDouble enzyme products were recovered by agarose gel electrophoresis,and the target gene and pEGFP-N 1 vector were recovered.After the transformation,a single colony was selected and identified as HIV-1 nef gene by PCR and double enzyme digestion.The results of sequencing were correct compared with HXB2,which proved that the eukaryotic expression vector of pEGFP-N1-nef was constructed successfully.3.Expression of HIV-1 Nef protein in U87 and SH-SY5Y cells and its effect on cell activityAfter eukaryotic expression vectors of pEGFP-N1-nef from H-TC,N-TC,H-SPL and N-SPL were transfected into U87 and SH-SY5Y cells for 12h,green fluorescence was observed in both cells.The results of western-blot 48 hours after transfection showed that there were specific bands at about 27 KD in each experimental group,which was consistent with the expected size of Nef protein.No bands were found in empty plasmid transfection and blank control.It was proved that Nef protein was successfully expressed in U87 and SH-SY5Y cells.The results of CCK-8 showed that Nef protein from H-TC,N-TC,H-SPL and N-SPL had no significant effect on the activity of U87 cells(P>0.05),but significantly reduced the activity of SH-SY5Y cells(P<0.05),and there was no significant difference between different experimental groups.4.Effect of HIV-1 Nef protein on autophagy of U87 and SH-SY5Y cellsIn U87 cells,compared with blank and empty plasmid control,the expression of LC3-II protein in H-TC,N-TC,H-SPL and N-SPL cells increased significantly(P<0.05).Moreover,LC3-Ⅱ protein in the CNS derived H-TC and N-TC groups was significantly higher than that in the peripheral derived H-SPL and N-SPL groups(P<0.05).The expression of p62 protein rised,but there was no significant difference between the groups(P>0.05).In SH-SY5Y cells,the expression of LC3-Ⅱ protein in H-TC,N-TC,H-SPL and N-SPL groups was significantly increased compared with the blank control and the empty plasmid control(P<0.05),and there was no significant difference between the groups.The expression of p62 protein rised,but there was no significant difference(P>0.05).5.Effect of HTV-1 Nef protein on apoptosis of U87 and SH-SY5Y cells(1)Apoptosis observed by DAPI stainingIn U87 cells,48h after transfection cells with pEGFP-N1-nef,there was no significant change in the nucleus compared with the control group.In SH-SY5Y cells,48h after transfection of pEGFP-N1-nef into SH-SY5Y cells,the nuclei were concentrated and shrunk,and the chromatin was aggregated,showing the nuclear characteristics of typical apoptotic cells.(2)Detection of caspase-9 and caspase-3In U87 cells,compared with blank and empty plasmid control,there was no significant difference in the expression of caspase-9 and caspase-3 in Nef group(P>0.05).In SH-SY5Y cells,compared with blank control and empty control,the expression of caspase-9 and caspase-3 in Nef group increased significantly.The difference was statistically significant(P<0.05).However,there was no significant difference between groups of Nef protein from different sources(P>0.05).CONCLUSION1.There are amino acid site variations in Nef protein from central and peripheral sources in HAD patient and non-HAD patient.2.Nef protein from H-TC,N-TC,H-SPL and N-SPL had no significant effect on the activity of U87 cells.It can inhibit the late stage of autophagy of U87 cells,but has no effect on apoptosis.3.Nef protein from H-TC,N-TC,H-SPL and N-SPL can reduce the activity of SH-SY5Y cells.It is suggested that Nef has toxic effect on SH-SY5Y cells.It can inhibit the late stage of autophagy and induce apoptosis.