Expression And Verification Of MiRNAs And Target Genes In HC-? With Over-expression And Low-expression Of SHOX

Objective: To establish the model of SHOX gene overexpression or interference in chondrocytes and to screen the differentially expressed miRNAs.The target genes that might be regulated by differential miRNAs were searched to provide new theoretical basis in SHOX and growth plate abnormalities.Methods:(1)Lentivirus was used to overexpress/interfere the SHOX expressionin Hc-? cells,and positively screened by puromycin.(2)Real-time PCR detected the relative expression of SHOX mRNA in each group.(3)miRNA microarray was used to analyze the differentially expressed miRNAs in the SHOX overexpression / interfere and non-treated groups.(4)Select the differential miRNA with opposite expression trend in the treatment group as the research object,and use Real-time PCR to verify the consistency.(5)TargetScan was used to predict target genes of miRNAs,and mimics of differential miRNAs were transfected in HC-a cells to detect changes in target gene expression.Results:(1)Successful screening of positive HC-a cells infected with lentiviral vectors.(2)The expression of SHOX mRNA was detected by Real-time PCR.Compared with the untreated group,the expression of SHOX mRNA in the over-expression group was up-regulated(P<0.001).The expression of SHOX mRNA in the interfering group was down-regulated,and the difference was statistically significant(P<0.001).There was no significant difference in the expression of SHOX mRNA between the empty control group and the untreated group(P>0.05).(3)Compared with the untreated group,425 miRNAs in the SHOX gene overexpression group were differentially expressed;compared with the untreated group,379 miRNAs in the SHOX gene low expression group were differentially expressed.(4)The expression levels of 6 mi RNAs in the treatment group was opposite: miR-126-3p,miR-192-5p,mi R-370-3p,miR-432-5p,miR-3162-5p,miR-4745-5p.Real-time PCR showed that the expression of miR-126-3p,miR-192-5p,miR-3162-5p,and miR-4745-5p were all significant.(5)Further studies with significantly different miR-3162-5p were performed.TargetScan online predicts STAT5 b as its target gene.After transfection of miR-3162-5p mimic in HC-a cells,the protein expression of STAT5 b was significantly reduced.Conclusion: Taken together,the present study shows that miR-3162-5p has a significant difference in SHOX gene over/interfere expression in chondrocytes.Further mechanistic analysis showed that miR-3162-5p may target STAT5 b and reduce its expression.The biological function of this mechanism needs further study.