| background:At present,there are about 30 million asthma patients in our country,30 million years of CARE study,over the age of 14 in eight provinces in China survey population prevalence was 1.24%,the process of global industrialization and urbanization,environmental pollution and climate change,the influence of such factors as the prevalence of asthma has a tendency to increase.Asthma is a chronic respiratory disease,need long-term drug control,the burden of disease is characterized by loss of labor lead to social and economic burden increase,is also on the consumption of health resources.Therefore,the study of the pathogenesis of asthma increasing breadth and depth,with the prevention and treatment of bronchial asthma has very important practical significance.Micrornas are a kind of endogenous single small rnas that length of about 20 to 25 nucleotides,coding protein,mature,in the body induced by combining zRNAz silence complex(zRNA-inducedzz silencingzz complex,zRISCz)after the transcriptional regulation of gene expression level.Micrornas through targeted mRNA play a role of transcriptional gene regulation after.Micrornas involved in a variety of fundamental biological processes,micrornas disorders can lead to the organic pathology.Micrornas in lung development and lung diseases such as asthma,chronic obstructive pulmonary disease,cystic fibrosis and the role of pulmonary hypertension in recent years also gradually began to study involved.Inflammation is an important pathological process in the development of asthma,the miRNA through targeted immune receptors and cytokines involved in regulating immune responses and inflammation.In the continuous in-depth study,the role of micrornas in asthma and the mechanism to help people know more and more,may be in the future of asthma diagnosis and assessment of prognosis and treatment of new targets,bring new hope for the diagnosis and treatment of asthma.ObjectiveScreening of miRNAs related to the onset of asthma and salmeterol drug therapy to explore it's role in clinical application and disease pathogenesis.Methods1.mouse model of acute asthma was constructed.The miRNA microarray was used to detect the miRNA expression profiles of lung tissues.Bioinformatics analysis was performed.Cluster analysis,target gene prediction,GO and pathway analysis were used to screen disease-associated miRNAs.The differentially expressed miRNAs were clearly verified by RT-PCR.Serum from control and asthma patients was collected and miRNA expression levels in peripheral blood were measured by RT-PCR.2.On the basis of studying the establishment of an acute asthma mouse model in the previous step,an asthmatic mouse model of salmeterol drug intervention was established to evaluate the comprehensive status of the model.At the same time,the miRNA microarray technique was used to analyze the miRNA expression during asthma attack and drug intervention.Spectral changes were analyzed by means of differential expression analysis and trend analysis of microarrays to screen for miRNAs that may be associated with the onset of asthma and salmeterol drug therapy;and their regulatory effects were verified by cell function experiments.Results1.Establishment of a mouse model of acute asthma and salmeterol drug intervention model,observed from general conditions,determination of pulmonary function airway responsiveness,pathological inflammatory changes,mucus secretion status,lavage fluid cell sorting test,and serum and lavage The IgE content of the liquid was evaluated comprehensively in terms of model construction and both models were successfully constructed.2.There were 854 different miRNAs in the asthma model group compared with the control group,with 155 significant ones.There were 48 miRNAs with a difference ?2,of which the most obvious one was miR-208A-5P,down 6.41.Times.Through the intersection of TargetScan and Miranda databases,8689 candidate target genes were predicted and the target gene range was optimized to 940.GO and KEGG analysis was performed.118 gene annotations and 43 signal pathways were obtained,which were involved in the regulation of cytokine interactions.Chemokine signaling pathways,leukocyte transendothelial movement,vascular smooth muscle contraction,cell adhesion,and endothelial hyperplasia and other biological processes,most of which are related to the occurrence and development of asthma diseases.Further validation of the 6 miRNAs with significantly different expression showed that miR-208a-5p,miR-133a-3p,miR-486-3p,miR-193b-3p,and miR-205-5p expression differences and miRNAs Consistent with the chip data,miR-92a-2-5p has an upward trend in the model group group compared with the control group,which is consistent with the trend of the chip data,confirming the reliability of the chip data.3.Detection of the miR-208A-5P and miR-133A-3P expression levels in peripheral blood of asthmatic patients revealed that abnormal expression was also found and could assist asthma diagnosis.4.miRNA microarray analysis of asthma model mice after intervention with salmeterol drugs.Through the screening of differential genes and trend analysis,we found that compared with the abnormally expressed miRNAs in the asthma group,there were 233 after salmeterol drug intervention.The expression of miRNA was reversed,and the expression difference was analyzed to be 1.5,P<0.05.Three drug-related miRNAs were obtained: miR-1291,miR-205-5p,and miR-142-5p.RT-PCR was performed and two of them were verified.The expression levels of miRNAs miR-1291 and miR-205-5p were decreased in the lung tissue of asthmatic mice and increased in the salmeterol drug intervention group,which was close to the control group,suggesting that miR-1291 and miR-205-5p participated in the study.Salmeterol treats the pathophysiological process of asthma.5.Application of house dust mite(Der p2)to stimulate airway epithelial cells found that application of miR-1291 mimics can increase the content of miR-1291 in airway epithelial cells stimulated by Der p2,and inhibit mucus synthesis.Conclusions1.Two mouse models of the acute asthma model and the salmeterol drug intervention model were successfully established.2.Successfully establish the miRNA expression profile of the mouse model of acute asthma,and through bioinformatics analysis,found that the miRNAs differentially expressed in the asthma model participate in many important biological processes of asthma,affecting many of the important signaling pathways.It plays a regulatory role in the development of asthma.3.miRNA expression profile analysis and RT-PCR validation confirmed by miR-208A-5P,miR-133a-3P,miR-486-3p,miR-193b-3P,miR-205-5p was significantly differentially expressed in the lungs of asthmatic mice.4.MiR-208A-5P and miR-133a-3P expression in peripheral blood of asthma patients decreased.Combined detection of peripheral blood serum levels may be a new auxiliary molecular marker for early diagnosis of asthma.5.Three drug-related miRNAs were obtained through differential analysis of the expression profiles of the three miRNA microarrays.RT-PCR assays were performed to verify that two of the miRNAs,miR-1291 and miR-205-5p,were found in lung tissues of asthmatic mice.The level of expression decreased,and the expression level in the salmeterol drug intervention group rose to close to the control group,which may be involved in the pathophysiological process of salmeterol in the treatment of asthma.6,Application of miR-1291 mimics can increase the content of miR-1291 in airway epithelial cells stimulated by Der p2,and inhibit the synthesis of mucus. |
PDF Full Text Request