Study On The Relationship Between MiR-21and TNF-α Expression In Alveolar Macrophages Induced By LPS

Objective:To observe the dynamic expression of miR-21and tumor necrosis factor alphaduring inflammatory reaction in alveolar macrophages induced by lipopolysac-charide, analysis of their possible relationship over time, and investigate the possiblerole of miR-21during inflammatory reaction in it.Methods:NR8383alveolar macrophages were seeded in6well plates in vitro at2×106cells/mL，90min later adherent cells were stimulated with LPS at1μ g/mL finalconcentration, supernatant and cells were collected after centrifugation at0h,3h,6h,12h time point. Expression of NF-κ B p65in the nucleus was determined by westernblotting, TNF-α mRNA and miR-21level in cells was detected by qRT-PCR, TNF-αprotein in the supernatant was measured by ELISA, correlation between TNF-αmRNA and miR-21was analyzed by Pearson correlation analysis.Results:(1) In NR8383alveolar macrophages treated with LPS, TNF-α mRNAaccumulated, peaking at3h(65.41±23.92fold，P＜0.01)，six hours later decreased to27.72±5.16fold (P＜0.05) and12.02±2.61fold(P＜0.05) at12h. Levels of TNF-αprotein in the culture supernatants increased at3h after LPS stimulation(379.37±63.81pg/mL，P＜0.01), continues to rise at6h(591.73±48.18pg/mL, P＜0.01), peak at12h(761.29±54.71pg/mL，P＜0.01).(2) Treatment of NR8383alveolar macrophages with LPS induced miR-21expression level gradually increased, expression of miR-21had no statisticsignificance between0h and3h,but gradually decreased at6h（3.27±0.54fold，P＜0.01）and12h(6.28±0.95fold，P＜0.01).(3) Treatment of NR8383alveolar macrophages with LPS induced NF-κBactivation, compared with the0h group, NF-κB-p65protein expression in thenucleus was up-regulated at3h,6h,12h time point. (4)There was a negative correlation between miR-21and TNF-α mRNA levelsin NR8383alveolar macrophages(r=-0.895，P＜0.01).Conclusion:In NR8383alveolar macrophages stimulated with LPS, miR-21expressionlevel gradually increased but TNF-α mRNA expression level gradually decreased, sothere show a negative correlation between miR-21and TNF-α mRNA levels,predicting that miR-21may negatively regulate inflammatory responses ofNR8383alveolar macrophages inflammatory responses.