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Objective To study the regulatory role of microRNA-16 (miR-16) on human pulmonary surfactant associated protein (SP).Method Human alveolar epithelial A549 cells were transfected by miR-16 analogue,analogue negative control,inhibitor and inhibitor negative control.Blank control group was also set up at the same time.The proliferative abilities of the cells in each group were measured using cell counting kit-8 (CCK8) test.The expressions of miR-16,SP-A,SP-B and SP-C mRNA were examined using reverse transcription polymerase chain reaction (RT-PCR).The protein levels of SP-A,SP-B and SP-C were examined using western blotting method.Result RT-PCR showed that the expression of miR-16 after transfected with miR-16 analogue (34.11± 1.79) was higher than the negative control group (1.65 ± 1.07) and the blank control group (1.07 ±0.50).The expression of miR-16 after transfected with miR-16 inhibitor (0.36±0.05) was lower than the negative control group (0.96±0.13) and the blank control group (1.05±0.20).The differences were significant (all P＜0.05),and indicated that miR-16 over-expression and suppression were successfully achieved.Compared with the blank control group,cell proliferation at different time points in the analogue negative control group and the inhibitor negative control group showed no significant differences (all P＞0.05).Compared with the blank control group (1.02±0.19,1.01±0.09,1.01± 0.12) and the analogue negative control group (1.08±0.24,1.00±0.14,1.00±0.05),miR-16 down-regulated the mRNA expressions of SP-A,SP-B and SP-C (0.58±0.16,0.67±0.05,0.61±0.12).On the other hand,compared with the blank control group (1.02±0.19,1.01±0.09,1.01±0.12) and the inhibitor negative control group (1.05±0.22,0.99±0.13,0.98±0.10),miR-16 up-regulated the mRNA expressions of SP-A,SP-B and SP-C (1.66±0.33,1.29±0.11,1.23±0.12)(all P＜0.05).The trends of protein level of SP-A,SP-B and SP-C were related to their mRNA expression.Conclusion This study indicates that miR-16 inhibits pulmonary surfactant associated protein in A549 cells.
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Objective To study the effect of small interfering ribonucleic acid (siRNA) silencing apoptosis signal-regulating kinase 1 (ASK1) on inflammatory response of lipopolysaccharide-induced alveolar epithelial A549 cells and its mechanism.Method Cell inflammation model of A549 cells was induced by lipopolysaccharide.The expression of ASK 1 in A549 cells was silenced by liposome transfection of siRNA.The mRNA and expression levels of ASK1,interleukin 6 (IL-6),interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α) in A549 cells were detected by immunoblotting,real-time fluorescence quantitative polymerase chain reaction and enzyme-linked immunosorbent assay.Result The expression of IL-6,IL-8 and TNF-α in the experimental group was significantly higher than that in the control group (P＜0.001),which indicated that the inflammatory model of A549 cells was successfully constructed.The mRNA level and expression of ASK1 in the interference group was significantly lower than that in the negative control group and the blank control group (P＜0.01),indicating that silencing ASK1 was also successful.The expressions of IL-6,IL-8 and TNF-α in the interference group (0.37±0.04,0.32±0.04,0.48 ±0.13) were significantly lower than those in the negative control group (1.04±0.11,1.22±0.19,0.93±0.14) and the blank control group (1.01±0.14,1.01 ±0.23,1.02±0.25).The expression of IL-6,IL-8 and TNF-α protein in the interference group (pg/ml) (122.6± 11.0,537.2±42.4,159.2± 19.6) were also significantly lower than those in the negative control group (267.4±20.4,1 289.8±55.3,327.0±26.3) and blank control group (246.6±18.7,1 300.3±35.6,325.2± 18.3),with significant difference (P＜0.05).There was no significant difference in each value between negative control group and blank control group (P＞0.05).Conclusion Silencing ASK1 by siRNA can down-regulate the expression of IL-6,IL-8 and TNF-α in A549 cells,suggesting that ASK 1 may be involved in the regulation of lipopolysaccharide-induced inflammation in A549 cells.
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Objective To investigate the role of signal transducer and activator of transcription 3 (STAT3) on the expression of surfactant proteins (SP) in alveolar epithelial cells line A549.Methods STAT3 overexpression lentivirus vector was constructed and transfected into A549 cells.Three small interfering RNAs (siRNA) were chemically synthesized and transfected into A549 cells by Lipofectamine 3000 to construct cells that silenced STAT3.The expression of STAT3,SP-A,SP-B,SP-C and SP-D were detected by Real-time PCR and Western blot.Results In A549 cells,over-or under-regulation of STAT3 were constructed successfully.When STAT3 was rendered over-expressed,the expression of SP-A,SP-B,SP-C,SP-D mRNA was significantly increased compared with Mock group(P ＜ 0.05).The proteins were found to be significantly increased as well.By contrast,when STAT3 was under-expressed,SP was down-regulated (P ＜ 0.05).Conclusion STAT3 regulates the expression of pulmonary surfactant proteins in A549 cells.Over-expression of the STAT3 gene promotes the expression of SP,and its under-expression inhibited it.
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Objective To study the roles of miR-20a in lipopolysaccharide induced inflammation of A549 cells and the possible mechanisms.Method The miR-20a mimic/inhibitor were transfected into A549 cells, and the cells were stimulated using lipopolysaccharide for 24 h.Interleukin-6 ( IL-6) and IL-8 were detected at mRNA level and protein level using real-time PCR and ELISA method , respectively.Protein expression of apoptosis signal regulating kinase 1 (ASK1)、P38、P-P38、JNK and P-JNK were detected using Western blot. Result Compared to mimic negative control group , the levels of mRNA and protein expression of IL-6 and IL-8 in the mimic group were all significantly decreased ( P<0.05).Compared to inhibitor negative control group , the levels of mRNA and protein expression of IL-6 and IL-8 in the inhibitor group were all significantly increased (P<0.05).The levels of ASK1, P-P38 and P-JNK protein in the mimic group were significantly lower than the mimic negative control group (P<0.05);the level of protein expression of ASK1, P-P38 and P-JNK in the inhibitor group were all higher than the inhibitor negative control group (P<0.05).Conclusion The regulation of ASK1 by miR-20a may play an important role in the inflammation process of acute respiratory distress syndrome .