Effect of miR-20a on pulmonary surfactant synthesis of alveolar epithelial cells A549 and its mechanism / 中华实用儿科临床杂志

ABSTRACT

Objective To explore the role of miR-20a on pulmonary surfactant synthesis of alveolar epithelial cells A549 and its potential mechanism.Methods Lentivirus miR-20a overexpression vector(miR-20a group) or lentivirus no-load vector(no-load group) was transfected into A549 cells,and the expression of green fluorescent protein(GFP) was observed to determinate the transfection effficiency;cell proliferation was detected by using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT);the bioinformatics software and database were applied to predict and analyze the target genes of miR-20a about lung development;expressions of miR-20a,pulmonary surfactant-associated protein A(SP-A),pulmonary surfactant-associated protein B(SP-B),pulmonary surfactant-associated protein C(SP-C) and pulmonary surfactant-associated protein D(SP-D) mRNA were detected by using quantitative real-time PCR(qPCR);the expressions of SP-A protein,SP-B protein,SP-C protein,SP-D protein and protein signal transducers and activators of transcription 3 (STAT3) were detected by using Western blot.Results Observation of GFP expression under a fluorescent microscope indicated similar transfection efficiency,and real time-PCR showed that the expression of miR-20a increased after being transfected with lentivirus miR-20a overexpression vector(3.85 ± 0.18)compared with the normal group (0.99 ± 0.04)and the no-load group (1.21 ± 0.12),and the differences were significant(t =10.85,9.64,all P ＜0.001).As a result,lentivirus miR-20a overexpression vector was constructed successfully.Online software predicted that STAT3 gene was likely to be the target gene of miR-20a.Compared with the normal group (24 h,48 h,72 h:0.23 ± 0.01,0.39 ± 0.01,0.56 ± 0.03) and the no-load group (24 h,48 h,72 h:0.25 ± 0.01,0.44 ± 0.05,0.59 ± 0.01),miR-20a did not change the cell proliferation at different time points(24 h,48 h,72 h:0.26 ± 0.01,0.41 ± 0.02,0.58 ± 0.02) (all P ＞ 0.05).Compared with the normal group (1.00 ± 0.05,1.24 ± 0.20,1.31 ± 0.09,0.89 ± 0.12) and the no-load group (0.76 ± 0.10,1.31 ± 0.13,1.50 ± 0.11,1.01 ± 0.11),miR-20a up-regulated the mRNA expressions of SP-A,SP-B,SP-C and SP-D (2.05 ± 0.17,2.14 ± 0.10,2.84 ± 0.09,1.66 ± 0.08),and the differences were significant (all P ＜ 0.05).Compared with the normal group (0.46 ± 0.01,0.27 ± 0.03,0.69 ± 0.01,0.43 ± 0.01) and no-load group (0.43 ± 0.01,0.21 ± 0.01,0.79 ± 0.02,0.44 ± 0.02),miR-20a also increased the protein expressions of SP-A,SP-B,SP-C and SP-D (0.55 ±0.01,0.47 ±0.05,0.96 ±0.02,0.59 ±0.03),the diffe-rences were statistically significant (all P ＜0.05).The expression of STAT3 in miR-20a group(0.37 ±0.05) was significantly lower than that in the normal group(0.60 ±0.04) and the no-load group (0.68 ±0.06),and the differences were statstically significant (all P ＜ 0.05) in A549.Conclusions STAT3 is a downstream target gene of miR-20a.miR-20a can promote pulmonary surfactant synthesis of alveolar epithelial cells A549 by inhibiting STAT3.