The Expression Of MicroRNA-646in A549Cell Treated By Lipopolysaccharide And Its Function Analysis

BackgroundAcute respiratory distress syndrome (ARDS) is by sepsis, multiple trauma, severe pneumonia and other diseases invaded massive release of inflammatory cytokines, clinical syndrome of alveolar epithelial cell injury and pulmonary surfactant synthesis decreased as the main performance. Its clinical features, including tachypnea and distress, progressive hypoxemia, X-ray showed diffuse alveolar infiltrates. Treatment method of ARDS is in increasing, but it still one of the important reasons for the threat to human life in the PICU. In the United States, there are1.9million patients with ARDS.750,000patients died due to ARDS, the mortality rate of up to39.4%. In china, the higher mortality of ARDS, reached71.4%. At this stage of ARDS pathogenesis is not yet fully understood, but you can clear pulmonary surfactant synthesis is reduced in ARDS. Schmidt found that in the bronchoalveolar lavage fluid of patients with ARDS surfactant associated protein A (SP-A)、surfactant associated protein B(SP-B)、surfactant associated protein C(SP-C)、dipalmitoyl phosphatidylcholine significantly reduced.With the improvement of the ARDS, a corresponding increased of their amount. In severe ARDS patients given SP-C can significantly alleviate the symptoms of hypoxia, and reduce mortality.microRNA (miRNA) is a group is widely present in eukaryotes but short non-protein-coding sequence RNA.Recent studies have found that miRNA has a wide range of gene regulatory function, it can regulate cell growth, differentiation, apoptosis and critical protein secretion. Multiple miRNA expression in one cell makes the cells in the environment of an inner-miRNA, which controls the thousands of gene encoding the mRNA. the expression of various proteins in a suitable level, regulation of eachkind of a key protein expression.At this stage there is a growing emphasis on the study of the relationship of miRNA and lung disease.Has been studied the miRNA and pulmonary Growth. pulmonary fibrosis, lung cancer, pulmonary inflammation aspects of the relationship, confirmed miRNA involved in the above-mentioned the activities. ARDS pathogenesis is not yet fully understood and the lack of effective treatment. miRNA role in the development process of ARDS, more and more attracted attention.miRNA mechanism may be an important complement to the gene, the protein levels of ARDS/ALI pathogenesis and treatment. Rarely involved in the pathogenesis of ARDS reported miRNA.What changes in miRNA levels in ARDS? Through the miRNA microarray injury in before and after the A549cells, we found that a large number of differentially expressed miRNAs, suggesting that miRNA may play a role in the occurrence of ARDS development process. We predict that the differential expression of miRNA target genes and found that the growth and development of the target gene and pulmonary surfactant protein, lung inflammation, apoptosis, tumor aspects related. We chose miR-646for following experimental and further explore the changes of miR-646in experimental group, predicted target genes of miR-646, to further improve the ARDS pathogenesis and provide a new target for the treatment of ARDS. Objectives:1. To detect the expression of SP-A、SP-C in A549cells treated by lipopolysaccharide (LPS)2. MiRNA expression spectrum is determined by microarray chip technology measured of LPS-treated A549cells, the following experiment is determined differentially expressed miRNA and its target genes3. Explore miRNA involved in the possible mechanism of ARDS development process, looking for new targets for the treatment of ARDS.Methods:1.A549cells were divided into experimental and control groups. A549cells of control group were treated with RPMI-1640and A549cells of experimental groups were treated by LPS(5μg/ml、10μg/ml、15μg/ml) in a duration of24hours.Immunocytochemical method and western blot were used to detect the change of SP-A、SP-C.QPCR was used to detect the change of SP-A、SP-C mRNA.2. The expression of miRNAs were detected by using miRNAs array in different groups. Identify the key differences in the expression of miRNAs. Bioinformatics software predicts key differentially expressed the largest miRNA target genes, to find a target gene protein that was play an important role in the process of development of ARDS, determine the target miRNA and its target genes. And real-time quantitative PCR method to detect miRNA expression in target cells in each group, to verify the accuracy of the gene chip.miRNA expression and target gene expression in correlation analysis was done.3. Verify the role of miRNA regulation of protein in vitro. A549cells were divided into five groups, mock group, miRNA mimics, miRNA mimics control group, miRNA inhibitor group, miRNA inhibitor control group.Added of miRNA Mimics when A549cells transfected with mock group without transfection agent; other group transfected A549cells were added miRNA mimics, miRNA mimics control, miRNA inhibitor, miRNA inhibitor control. To do real-time quantitative PCR validation of miRNA expression in target cells in each group24hours to verify the effect of transfection transfection. Finally, western blot to detect the protein expression of target genes in the cells in each group.Results:1. The expression of SP-A, SP-C in each group1.1Before LPS treatment, the morphology of A549cells polygonal, plump cytoplasm, cytoplasmic processes were adherent growth the overgrown integrated into the film, the non-nuclear pyknosis phenomenon. A549cell morphology round, the volume was reduced and the cytoplasm becomes empty bright, and intracytoplasmic particles of different sizes,24h after LPS treatment.1.2Detect the A549cells SP-A and SP-C expression levels using immunocytochemical staining. SP-A in the relative expression level of the control group was0.0754±0.0009,5μg/ml LPS treatment group relative expression level was0.0707±0.0019,10ug/ml LPS treatment group relative expression level was0.0424±0.0028,15μg/ml LPS treatment group relative expression level was0.0155±0.0019. Experimental group and the control group, the difference was statistically significant (F=534.498, P<0.05). SP-C in the relative expression level of the control group was0.0508±0.0035,5μg/ml LPS treatment group relative expression level was0.0268±0.0035,10p.g/ml LPS treatment group relative expression level was0.0148±0.0025,5μg/ml LPS treatment group relative expression level was0.0226±0.0035.The experimental group and the control group, the differences were statistically significant (F=68.388, P<0.05).1.3Detect the A549cells SP-A and SP-C expression levels using western blot Compared with control group, the expression of SP-A in cytoplasm of A549cells was decreased in experimental groups (P<0.05), lipopolysacchride at different concentrations induced the expression of SP-A in A549cells in a dose-dependent manner. Compared with control group,the expression of SP-C in cytoplasm of A549cells was decreased in experimental groups (P<0.05).The expression of SP-C was the lowest in10mg·L-1lipopolysacchride treatment group.1.4Detect the A549cells SP-A and SP-C expression levels using QPCR Relative expression level of SP-A mRNA in the control group was1.0509±0.0368,5μg/ml LPS treatment group relative expression level was0.9333±0.0146,10μg/ml LPS treatment group relative expression level was0.5630±0.0097,15μg/ml LPS treatment group relative expression level was0.2636±0.0248. The experimental group and control group differences were statistically significant (F=676.753, P <0.01). SP-C mRNA relative expression level in the control group was1.0442±0.1559,5μg/ml LPS treatment group relative expression level was0.5039±0.0175,10μg/ml LPS treatment group relative expression level was0.2306±0.0574,15μg/ml LPS treatment group relative expression level was0.3987±0.0084. The difference of the experimental and control groups were statistically significant (F=21.635, P<0.01).The above results show that the cells SP-A, SP-C expression in the experimental group were decreased compared with the control group, the difference was statistically significant (P<0.05). These are consistent in ARDS.These results and in ARDS, SP-A, SP-C expression is consistent.2. Gene chip results and determine the target miRNA、target genes2.1miRNA microarray results:31miRNA expression was significantly up-regulated, which express the greatest difference was miR-646up to3.4times, miR-1247-3p up to3.2times.13miRNA expression was significantly down-regulated, which express the greatest difference was miR-4455down to0.32 times, miR-374b-5p down to0.36times. Differentially expressed miRNA target gene prediction using bioinfonnatics software (TargetScan, miRanda, Clip-seq. miRDB).4software predicted miR-646a total of512target genes, including four software Total176target genes, sftpc is a candidate target gene in the total of the target gene, which encodes a protein is SP-C.SP-C to play an important role in the pathogenesis of ARDS.We identified the following experiment the target miRNA is miR-646, the the goal target gene protein is SP-C.2.2Detect the expression of miR-646expression using QPCR The relative expression level of the control group of miR-646was0.9597±0.020,5μg/ml LPS treatment group was1.6642±0.0938,10μg/ml LPS treatment group was2.4762±0.1380,15μg/ml LPS treatment group was1.6319±0.1325. Experimental group and control group differences were statistically significant (P<0.05),10ug/ml group relative expression level is highest.. The above results and miRNA microarray results were consistent, to verify the accuracy of the chip.2.3Correlation Analysis:In LPS treatment of A549cells, the expression levels of miR-646and SP-C expression levels were significantly correlated (P<0.05), and showed the concentration-dependent negative correlation (pearson correlation coefficient r=-0.916).3. Verify in the role of the miR-646of the SP-C in vitro3.1We observed see cytoplasmic green fluorescence with a fluorescence microscope at48hours transfection.Real-time quantitative PCR:relative expression level of miR-646in the mock group was256.85±90.12, relative expression level of miR-646in miR-646mimics group was194215.66±12555.57, relative expression level of miR-646in miR-646Mimics control group was145.99±22.40, relative expression level of miR-646in miR-646inhibitor group was1.04±0.23, relative expression level of miR-646in miR-646inhibitor control group was173.59±15.94. miR-646mimics group, miR-646inhibitor group and mock group difference was statistically significant (P<0.05).these results suggest that successful transfection.3.2To detect the expression of SP-C using western blotCompared with the mock group, the expression level of SP-C in miR-646mimics group was significantly decreased, the difference was statistically significant (P<0.05).Compared with the mock group, the expression of SP-C in miR-646inhibitor group was significantly increased, and the difference was statistically significant (P<0.05).Compared with the mock group, the expression of SP-C in miR-646mimics control group、miR-646inhibitor control group had no significant change, the difference was not statistically significant (P>0.05).Conclusions1. Expression levels.of SP-A, SP-C was decreased in A549cells after LPS treatment.These results and the clinical results are consistent.2. miR-646may play a biological function in acute respiratory distress syndrome according to inhibiting the transcription of SP-C.