Screening Of Differentially Expressed Proteins In HC-a Cells With SHOX Gene Overexpression Or Interferencce

ObjectiveTo screen for differentially expressed proteins in human articular chondrocytes(HC-a)with short stature homeobox-containing gene(SHOX)overexpression or interference.Methods1.To get the concentration of puromycin for selecting the positive chondrocytes infected with lentivirus vectors,different concentrations of puromycin were added into the chondrocyte culture medium,and chondrocytes were continued culture to observe the survival of chondrocytes.2.HC-a cells were infected with lentivirus containing SHOX overexpression vector,lentivirus containing SHOX shRNA interference vector,lentivirus containing overexpression empty vector and lentivirus containing interference empty vector,respectively.Positive cells were selected by puromycin.3.The expression level of SHOX mRNA was deteced by quantitative real-time PCR and the relative quantity of SHOX protein was determined by western blot.4.Three groups included overexpression group,interference group and untreated group of HC-a cells were chosen to perform isobaric tags for relative and absolute quantitation(iTRAQ)combined with liquid chromatography and tandem mass spectrometry(LC-MS/MS)analysis to explore the differentially expressed proteins.Proteins at 1.2-fold change and P <0.05 were chosen to be threshold for selecting differentially expressed proteins.5.Functional annotation and pathway analysis of identified differentially expressed proteins were performed by using the Gene Ontology(GO)database and the PANTHER classification system.Results1.The minimum concentration of puromycin at 2.5ug/ml caused all chondrocytes killed just after 3 days of culture with medium containing puromycin was chosen to select positive cells.2.Positive HC-a cells infected with each lentivirus vector were screened out,respectively.3.Compared with untreated group,the up-regulation of SHOX mRNA in overexpression group was statistically significant(P <0.001),and the down-regulation of SHOX mRNA expression in interference group was statistically significant(P <0.05).There were no significant differences in SHOX mRNA expression between the two empty vector control groups and the untreated group(P> 0.05).Compared with the untreated group,the expression level of SHOX protein in the overexpression group was increased,and the expression level of SHOX protein was decreased in the interference group.4.Compared with the untreated group,26 differentially expressed proteins were identified in the overexpression group,21 of them were up-regulated and 5 were down-regulated.91 differentially expressed proteins were identified in the interference group,of which 59 were up-regulated,32 were down-regulated.A total of 16 differentially expressed proteins were identified as in both the overexpression group and the interference group.Among them,12 proteins were changed in same trend,4 proteins were opposite.5.The 16 overlapped differentially expressed proteins mainly involved 5 kinds of cellular component,5 kinds of molecular function,10 kinds of biological process,15 kinds of protein class and 13 kinds of signal pathway.ConclusionsThe SHOX gene was overexpressed and interfered in HC-a cells by lentivirus vectors,respectively.The differentially expressed proteins among SHOX overexpression group,SHOX interference group and untreated group were selected out.The relationship between SHOX and the differentially expressed proteins needs further study.