Verification Of Sepsis-related Target Genes Regulated By MiRNA-223, MiRNA-15a And MiRNA-16 In THP-1 Cells

Objective:Sepsis is a systemic inflammatory response syndrome caused by infection, that features rapid disease progression, poor prognosis and high mortality. It is an important issue and research hotspot in clinic. Bioinformatics analysis indicated that FOXO1 and ACVR2A were the predictive target genes of miRNA-223, miRNA-15a and miRNA-16. In this study, we are trying to validate the target genes in THP-1 cell line predicted by bioinformatics analysis and lay the foundation for the further study of signaling pathway about these two genes.Methods:(1) The expression levels of miRNA-223/15a/16 and FOXO1/ACVR2A mRNA were evaluated by qRT-PCR analysis in THP-1 cell line induced by in LPS at each time point, respectively. (2) QRT-PCR was conducted to detect the expression levels of miRNA-223/15a/16 and FOXO1/ACVR2A mRNA 48 hours after transfection with miRNAs’mimics/inhibitors. (3) The protein levels of FOXO1 and ACVR2A were measured by Western Blot 48 hours after transfection.Results:(1) After THP-1 cells induced by LPS, the expression levels of miRNA-223, miRNA-15a and miRNA-16 were all down-regulated on the time point of 12h/24h/48h (P<0.01), then reached the lowest levels on the time point of 24h-48h and closed to the levels of miRNAs on Oh and 72h. while the mRNAs expression levels of FOXO1 and ACVR2A were all remarkedly increased at 24h/48h/72h (P<0.01). (2) In THP-1 cells, through transfection with miRNA Mimics, levels of miRNA-223/15a/16 were increased. The gene and protein levels of FOXO1 and ACVR2A became significantly decreased than those of their controls (P<0.01). (3) After transfection with miRNA Inhibitors, the relative levels of miRNA-223/15a/16 were decreased. The gene and protein levels of FOXO1 and ACVR2 A became significantly higher than those of their controls (P<0.01).Conclusion:FOXO1 can be regulated by miRNA-223, miRNA-15a and miRNA-16 in THP-1 cell line.