A Preliminary Study Of MicroRNA Expression Profile In LPS-induced ALI/ARDS Mouse Lung Tissue

[Background] Acute respiratory distress syndrome was first reported by Ashbaugh and his colleagues in1967, it was originally named adult respiratory distress syndrome, but it also appears in children, so the AECC renamed it as acute respiratory distress syndrome in1994. ALI/ARDS was a severe respiratory disease which did not secondary to cardiac disease. Because of the modern recovery technology and the improvement of early rescue for critically ill disease, the early mortality of critically ill patients reduced and the incidence of ALI/ARDS would increase, in the pediatrics intensive care unit (PICU), the morbidity is about1.0%～1.0%. Although the lung protective ventilation strategies and other modern treatments could improve the ventilate/blood flow disproportion and the oxygen supply to some extent, they did not reduce the mortality of ALI/ARDS, ALI/ARDS was still a threat to children’s life in the PICU, the mortality is as high as54.6%-62.9%.ALI/ARDS is based on diffuse lung cell injury, the pathology characteristics are pulmonary edema and inflammatory cells infiltrated into the lung tissue which is caused by pulmonary vascular injury, They show as severe hypoxemia, diffuse pulmonary infiltrates and pulmonary edema in clinical. The pathogenesis of ALI/ARDS is complicated, and it’s not yet entirely clear. But It’s reported that mainly involved in the following aspects:1. The formation of pulmonary edema:now the factors of the edema are generally accepted as the two aspects:on one hand, it is due to the permeability increase of the alveolar epithelium and the capillary epithelial, alveolar-capillary barrier destroyed, the fluid transfer function is impaired, it makes the edema fluid which rich in protein gathered in the cavity; On the other hand, the change of the amount and function of aquaporin contribute to this. Aquaporin mainly includes the AQP-1, AQP-3, AQP-4and AQP-5. It is accepted that the AQP1play an important role on the absorption and transport of the liquid in the lungs. In ARDS, AQP-1, which express in alveolar type II, decreased the reabsorption of the water by aquaporin, water gathered in the pulmonary interstitial and caused alveolar edema.2. The excessive inflammatory reaction in the lung tissue. The character of the ALI/ARDS is the excessive inflammatory response, much evidence show that polymorphonuclear leukocytes (PMN) play a key role in the pathogenesis of ALI/ARDS. PMN mediated oxidative stress, the release of proteolytic enzymes and the cytotoxin, all these caused the damage of pulmonary vascular endothelial and alveolar epithelial, large amounts of liquid which is riched in protein entered into the lung tissue, and leading to the formation of pulmonary edema, hyaloid membrane and alveolar collapse. In addition, the release of cytokines, proinflammatory factor and anti-inflammatory factor imbalances, all these were the important reasons for ALI/ARDS.3. The damage of alveolar epithelial type II reduced the synthesis of surfactants, also changed the composition of the surfactants. The substances formed a monolayer interface between the liquid and the gas, which would reduce the surface tension. The monolayer interface played an important role in maintaining the normal structure of the alveolar and stabilizing the alveolar ventilation. Therefore, the reduce of surfactants would cause alveolar collapse, hypoxemia and other serious symptoms.4. coagulopathy:It had been confirmed that coagulation and fibrinolytic cascade disorder were appeared in the development of ALI/ARDS. It was very common that ALI/ARDS patients had a pulmonary intravascular platelet fibrinous thrombus and cellulose composure. It believed that coagulopathy contributed to multiple organ failure. It also found that diffuse intravascular coagulation (DIC) associated with the severity of the disease. At present, there is no specific treatment for ALI/ARDS, all the treatments were based on its pathophysiological changes and clinical manifestations. Comprehensive treatment measures were taken, mainly including:1. Timely treatment of the primary disease and systemic nutritional support:it is the first principle that removes or controls the pathogenic factors as soon as possible to inhibit systemic inflammation. Systemic comprehensive support treatment, such as prevention of infection, nutritional support, protection of gastrointestinal tract, prevention of thromboembolism and so on can prevent disease progression; even can avoid the occurrence of multiple organ dysfunction syndromes (MODS).2. Treated with mechanical ventilation:ventilation therapy is the main methods to correct hypoxia. In recent years, ventilation therapy has made remarkable progress in the treatment of ALI/ARDS. Especially the lung protective ventilation strategy,(low tidal volume:6ml.kg-1) can effectively improve alveolar ventilation, PaO2and arterial oxygenation. The lung protective ventilation strategy also can avoid the occurrence of ventilator-induced lung injury.3. A number of drugs, such as exogenous lung surfactant (PS), glucocorticoids, protease inhibitors and expectorant drugs play an important role in improving the ventilate/blood flow rate and enhancing the blood oxygen. But these drugs did not reduce the mortality of ALI/ARDS patients.Lee discovered the first microRNA (miRNA) in caenorhabditis in1993, since then, miRNA has been a hotspot in many fields. In2002, the famous American magazine "Science" called it as the first one of the top ten scientific and technological breakthroughs. MiRNA, a kind of non-coding single chain small molecule, can cause degradation of the target mRNA, or inhibit its translation by complementary pairing with the nucleotide, then regulate the gene expression in a post-transcriptional way. MiRNA plays an important role in regulating the life process, involving the early development of cells, growth, differentiation, apoptosis, tissue development, and the key protein secretion etc. In recent years, studies have shown that miRNA involved in the pathology and physiological processes of many diseases, such as cardiovascular disease, tumorigenesis, inflammation and never disease. A large number of studies have shown that miRNA closely relates to the growth and development of lung and lung disease, such as lung cancer, pulmonary fibrosis, and pneumonia.Bio-chip is a new technology which is charactered by high-throughput, high integration, miniaturization, continuous and automated, it is originated form nucleic acid hybridization. The so-called bio-chip generally refers to microarray hybridization chips (micro-arrays) which the biometric information (such as gene fragments, DNA fragments or polypeptides, proteins, sugar molecules, tissue, etc.) were high density fixed to the medium. The sequence and position of every molecule in the array are known, they are pre-set sequence lattice. The miRNA microarray skills applied to the detection and comparison of normal tissue and diseased tissue expression profiling of miRNA. We can find their target genes according to bioinformatics analysis, and have a deeply understanding about their regulatory mechanism or biological functions, all these will help for the diagnosis and treatment of the diseases.Based on the above theory, this study intended to build a ALI/ARDS mouse model by intratracheal instillation of lipopolysaccharide, we evaluated whether the ALI/ARDS mouse model successed by observing general situation, the respiratory rate, wet and dry weight ratio of lung tissue and the histopathology. MiRNA expression profiles of ALI/ARDS mouse lung tissue are detected by microarray, and then the differentially expressed miRNA are screened. The target genes of differentially expressed miRNA prediction are using bioinformatics methods. After that, we will have an advanced analysis about the target genes. We believe that all these will provide new ideas for further completing the pathogenesis of ALI/ARDS and provide a new way for the treatment of ALI/ARDS.[Method]24C57BL/6mice were randomly divided into experimental and control groups,12in each group. The ALI/ARDS model mice were induced by injecting LPS (6mg·kg-1) into trachea. The mice of control group were injected with the same method with an appropriate dose of saline. All mice were sacrificed24hours after surgery. The general condition, respiratory rate, the ratio of wet and dry weight of the mice lung tissue, pathological changes of the lungs were observed to estimate whether the model successed or not. MiRNA microarray was used to detect miRNA expression profile in mice lung of ALI/ARDS group and the control group, and then the visible different miRNAs were chosen to have an advanced study. Four miRNA target gene prediction softwares (TargetScan, Miranda, Clip-seq, miRDB) were used to predict the target genes of the differently expressed miRNAs. We choose the genes as candidate target genes if three or more software have the same result. Then the predicted target genes had a Gene Ontology analysis (GO, http://www.geneontology.org) and the Gather KEGG analysis (Http://gather.genome.duke.edu) to predict the related signaling pathway.[Result]1、Compared with the control group, the experimental group mice showed lethargy, poor activity, lips cyanosis, shortness of breath, and the respiratory rate was significantly faster. Secondly, the ratio of wet and dry weight of lung tissue was significantly higher in the experimental group mice which indicating pulmonary edema. The last, lung histopathology results showed alveolar septum damage, inflammatory cell infiltration, all these coincided with the pathological changes of ALI/ARDS.2、The microarray results showed48miRNAs had significant different expression in mice lung tissue of ALI/ARDS,27of which were upregulated,including:miR-574-5p, miR-483-5p, miR-711, miR-30c-1-3p, miR-92a-2-5p, miR-709, miR-466j, miR-690, miR-1902, miR-1897-5p, miR-1940, miR-1894-3p, miR-3099-3p, miR-1249-5p, miR-149-3p, miR-193b-5p, miR-3087-5p, miR-3102-5p, miR-3473, miR-466m-5p, miR-504-3p, miR-5107, miR-5110, miR-5115, miR-5132, miR-669f-5p, miR-423-5p.21microRNA were downregulated, including miR-98-5p, let-7a-5p, miR-146b-5p, miR-200a-3p, miR-30b-5p, miR-145-5p, miR-130a-3p, miR-15a-5p, miR-467c-5p, miR-18a-3p, miR-133a-3p, miR-151-3p, miR-16-5p, miR-24-3p, miR-30d-5p, miR-34c-5p, miR-23a-3p, miR-3472, let-7g-5p, miR-3473b, miR-5128。3、Using the target gene prediction software, the number of the candidate target genes of let-7a-5p are267, miR-16-5p are46, miR-24-3p are374, and miR-146b-5p are137. GO analysis were used to enrich and classify the target genes to find out which related to inflammation, lung development and apoptosis, the result showed that48target genes of let-7a-5p associated with inflammation,16related to apoptosis and6target genes related to lung development;23target genes of miR-16-5p associated with inflammation,12related to apoptosis and4target genes related to lung development; as for miR-24-3p,20target genes associated with inflammation,7related to apoptosis, and17target genes related to lung development. Gather KEGG analysis was used to find out the most relevant biological pathways, these signaling pathways include MAKP signaling pathway, Toll-like receptor signaling pathway, TGF-p signaling pathway.[Conclusion] We successfully constructed the mice ALI/ARDS model by tracheal injection of LPS, miRNA have a significantly different expression profile in the lung tissue of ALI/ARDS mice model. The target genes of these differentially expressed miRNA involved in inflammation, lung development, and apoptosis, suggesting that miRNA play an important role in the pathophysiology of ALI/ARDS process.