Effect of miR-20a on pulmonary surfactant synthesis of alveolar epithelial cells A549 and its mechanism

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 efficiency; 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 0.05). 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 statistically 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. Key words: miR-20a; Pulmonary surfactant; Lung epithelial cells A549