MiR-21 Inhibitor Inhibits The Expression Of TNF-? In LPS-induced Alveolar Macrophages

Objective:Based on our previous research,we downregulated the level of miR-21 in rat alveolar macrophages by transfection of miR-21 inhibitor,and observed the effect of miR-21 on the lipopolysaccharide(LPS)-induced production of tumor necrosis factor alpha(TNF-?)in alveolar macrophages,and explored the possible regulation mechanism of miR-21 on the LPS induced alveolar macrophage inflammatory reaction.Method:1)We selected the rat alveolar macrophages(NR8383)as the research object,and transfected the NR8383 cell with 10 n M and 20 n M,40 n M concentration of the fam labeled micro RNA negative control for 24 hours respectively.We detected the miRNA transfection efficiency by immunofluorescence,and selected concentration which can offer the best transfection efficiency as the subsequent experiments transfection concentration;2)After the NR8383 cell were transfected with 20 n M miR-21 inhibitor or control negative for 24 hours respectively,we detected the level of miR-21 and the PTEN m RNA by RT-q PCR and the level of PTEN protein were detected by western blot.3)The rat alveolar macrophages were divided into two groups in vitro,and transfected with 20 n M miR-21 inhibitor or negative control for 24 hours.A final concentration of 1 ug / ml LPS was used to stimulate the NR8383 cells for 6 hours and then we collected the cells and cell culture supernatant;We detected the expression of p-Akt protein by Western blot and the immunofluorescence was used to detect activation of NF – ?B;The TNF-? m RNA expression levels in two groups were detected by RT-q PCR,and the TNF-? protein form the cell culture supernatant was detected by enzyme linked immunosorbent assay,(enzyme linked immunosorbent assay,ELISA)Result:1)We used 10 n M?20n M?40n M concentration of FAM labeled miRNA negative control to transfect the NR8383 cells for 24 hours,then determined the transfection efficiency by Immunofluorescence.the result showed transfection efficiency reached the highest point by 20 n M,So we chose the 20 n M as the transfection concentration in subsequent experiments.2)Compared with the NC transfection group,the level of miR-21 dropped to0.34 + 0.08 times(P < 0.05)in miR-21 inhibitor transfection group,the expression of PTEN m RNA increased by 2.24 + 0.48 times(P < 0.05)and PTEN protein expression increased by 3.47 + 0.98 times(P < 0.05)in miR-21 inhibitor transfection group.3)LPS stimulated cells in both groups for 6 hours,compared with the NC group,the expression of p-Akt protein in the miR-21 inhibitor transfected group decreased to(0.19 ±0.11)times(P < 0.05);and the nuclear translocation of NF-?B was significantly reduced;The expression of TNF-?protein form cell culture supernatant fell form(2760.50± 302.80)to(922.88± 68.58),dropped to 0.34 + 0.04 times(P < 0.05)and TNF-? m RNA expression levels dropped to 0.34 + 0.78 times(P <0.05).Conclusion:1)Downregulation of miR-21 can inhibit the expression of TNF-? in LPS-induced NR8383 alveolar macrophages.2)Downregulation of miR-21 inhibit the prouction of TNF-?by downregution of PTEN / AKT and the activation of NF-?B.