The Study Of Changes In Proteome Of HepG2 Cells Induced By Riboflavin Deficiency

ObjectiveTo establish a cell model of riboflavin deficiency,and then culture cells with different concentrations of riboflavin in order to observe the level of riboflavin that could maintain the cytoactive of cells,and to detect the proteomics changes of HepG2 cells induced by riboflavin deficiency,followed by further analyzing and verifying the results of proteomics.This study will provide scientific basis for further study on the mechanism of riboflavin deficiency on human body.Methods1.Removal of riboflavin in fetal bovine serum(FBS): Using the method of ultraviolet irradiation to remove riboflavin in fetal bovine serum on sterile condition.The levels of riboflavin concentrations in FBS with different irradiation time were measured by high performance liquid chromatography(HPLC).Cells were cultured in FBS with different irradiation time for 96 h to measure the cell vitality at different time intervals.And then we choose the irradiation time that can bring a low riboflavin concentration level in FBS,andmeanwhile maintain the cell viability during 96 h culture,which would be the pretreatment method of our next cell model building.2.The establishment of riboflavin deficiency cell model and the exploration of appropriate intervention concentration: by customizing riboflavin-free medium,UV irradiation removal of FBS riboflavin to establish riboflavin deficiency culture conditions.On this basis,cells were cultured with different concentrations of riboflavin(0.76 、 3.76 、 6.76 、 12.76 、 24.76 、48.76nmol/L)for 96 h.The cell viability and apoptotic rate were measured at different time points,and the content of malondialdehyde(MDA)in culture supernatant,the activity of extracellular aspartate aminotransferase(AST)and alanine aminotransferase(ALT),as well as intracellular glutathione reductase(GR),glutathione peroxidase(GSH-Px),superoxide dismutase(SOD)activity,glutathione(GSH)and oxidized glutathione(GSSG)were measured at 72 h.3.Proteomics technique to detect the effect of riboflavin deficiency on protein expression profile of HepG2 cells: Using label free quantitative proteomics techniques to analysis and compare the proteomic difference between the riboflavin deficient(0.76nmol/L)and riboflavin moderate(12.76nmol/L)group,followed by bioinformatics analysis(including GO enrichment analysis,KEGG pathway analysis,differential protein interaction network analysis).The four key differentially expressed proteins(NDUFS1,NDUFV2,SDHA,SQSTM1,ERO1A)were tested by Western blotting.4.Effects of riboflavin deficiency on apolipoprotein B100(ApoB100)expression pathway and disulfide bond formation in Hep G2 cells: The results of our proteomic study showed that riboflavin deficiency down regulated a key protein(ERO1)which could affect the folding of apolipoprotein B100 disulfide bonds.Intracellular and extracellularly secreting ApoB100 were measured by enzyme-linked immunosorbent assay(ELISA).The gene expression of ApoB100 was detected by reverse transcription-real-time fluorescence quantitative(RT-qPCR).The gene and protein expression of protein disulfide isomerase(PDI)were measured by RT-qPCR and Western blotting,respectively.Using Ellman’s method to determine the protein bounding mercapto in cells to indirectly reflect the ApoB100 disulfide bond formation level.Results1.The riboflavin content in FBS was decreased with the prolongation of irradiation time,and the content of riboflavin in FBS with only 30 min irradiation time was significantly decreased(P<0.01).As irradiation time continued prolonged,the decline trend of riboflavin concentration was gradually weakened.The viability of the cells cultured in the medium with30 min irradiation time serum had no significant difference with the serum without irradiation(P<0.05),and with the prolongation of irradiation time,the cell viability significantly decreased(P<0.05).3.During 72 to 96 h culture,as riboflavin concentration decreased,the viability of Hep G2 cells significantly decreased and the apoptosis rate significantly increased(P<0.05),the activity of AST and ALT in culture medium significantly increased and the content of MDA significantly increased(P<0.05),the activity of GR significantly decreased and the activity of GSH-Px significantly increased,the content of GSH significantly decreased and the content of GSSG increased significantly and GSH / GSSG decreased significantly(P<0.05).The breaking point of most of the above indicators were about 12.76nmol/L.It was proved that riboflavin-deficient cell model was established successfully,and the riboflavin concentration that could maintain normal cell status should be higher than 12.76nmol/L.3.A total of 3730 proteins were identified in this study,of which 2830 proteins were identified in the riboflavin deficiency group and 3020 proteins were identified in the control group,85 proteins were specific to the riboflavin deficiency group and 275 proteins were control.In the 2745 proteins shared by the two groups,37 proteins with different expression ratios greater than 2 were obtained after comparison.Among them,as compared to the control group,13 proteins were found significantly upregulated,while 24 proteins were significantly down-regulated in the riboflavin deficiency group.The results of GO enrichment analysis showed that 37 differentially expressed proteins were highly enriched in mitochondrial oxidative respiratory chain,and the molecular function was mainly redox activity.It was mainly involved in the biological processes such as electron transfer,redox,energy metabolism and so on.KEGG signal pathway analysis showed that these differential proteins were involved in18 signaling pathways,among which with higher matching rate were the pathways of Parkinson’s disease,fatty acid metabolism and endoplasmic reticulum stress.Western blot results showed that the expression of NDUFS1,NDUFV2,SDHA,ERO1 A was significantly up-regulated and the expression of SQSTM1 was significantly decreased in the riboflavin-deficient group compared with the control group(P<0.05),which was consistent with the proteomic result.4.Compared with the control group,the ApoB100 contents of inracellular and extracellular secretion significantly decreased in the riboflavin deficiency group(P<0.05).The gene and protein expression of PDI were both significantly lower in the riboflavin deficiency group.The gene and protein expression of GRP78 and GADD153 both significantly increased(P<0.05),and the total protein bounding mercapto in cells significantly decreased in the riboflavin-deficient group(P <0.05).ConclusionRiboflavin deficiency significantly affects the normal state of Hep G2 cells,and the concentration of riboflavin that could maintain the normal cell state should be higher than 12.76nmol/L.Riboflavin deficiency significantly affected the protein expression profile of HepG2 cells.The influenced proteins could down-regulate energy metabolism and lipid metabolism,and promote the development of endoplasmic reticulum stress and apoptosis.Further study of apoB100 pathway found that riboflavin deficiency could lead to ApoB100 disulfide bond formation disorder and then affect lipid transport.This study provides a clue and experimental basis for further exploring the molecular mechanism of riboflavin deficiency in human body.