| Respiratory distress syndrome(RDS)is caused by insufficient pulmonary surfactant(PS)synthesis in alveolar type II cells(AT-II).That is,a critical illness with progressive dyspnea and respiratory failure has a high incidence and mortality,which seriously affects the quality of life and prognosis of neonates.The main purpose of this experiment is to study the mechanism of neonatal respiratory distress syndrome,explore the synthetic process of PS,and hope to provide a new intervention target for the prevention and treatment of the disease.It has been reported that the synthesis and secretion of PS are influenced and regulated by many factors.Among them,the role of Micro RNAs(mi RNAs)has attracted more and more attention.mi R-20a-5p is a mi RNA screened by the gene chip technology in our previous research.The microarray results show that mi R-20a-5p is gradually downregulated with fetal age during the lung development.In addition,it was differentially expressed in peripheral blood of children with RDS and controls,and mi R-20a-5p was significantly downregulated in children with RDS.Therefore,we speculate that mi R-20a-5p may play a role in lung development and the development of RDS.This project was divided into two parts.In the first part,the differential expression of mi R-20a-5p was verified by real-time quantitative PCR,and bioinformatics analysis was performed to understand its basic biological characteristics.The second part was to explore the role of mi R-20a-5p in regulating the expression of PS in fetal alveolar type II epithelial cells and its mechanism.Part ?: Differential Expression and Basic Biological Characteristics of mi R-20a-5pPurpose: To observe the expression characteristics of mi R-20a-5p during fetal lung development.To observe the expression of mi R-20a-5p in plasma of children with RDS.To perform bioinformatics analysis of mi R-20a-5p,and to provide a theoretical basis for exploring the biological function of mi R-20a-5p in the development of lung.Approach: Real-time quantitative PCR was used to detect the expression of mi R-20a-5p in lung tissue of fetal rat at 16,19,and 21 days of pregnancy,and its expression in plasma of children with RDS and controls.The mi RBase was used to search for mi R-20a-5p sequence characteristics.The species conservation was analyzed.Targetscan and other databases were used to predict the target genes,then they were analyzed by gene ontology(GO)and pathway analysis.Results: The expression level of mi R-20a-5p gradually decreased in fetal lung tissue at 16,19 and 21 days of pregnancy.It was down-regulated in plasma of children with RDS compared with control children.The sequence of mi R-20a-5p is highly conserved among multiple species,and its target genes are present in various cell components.These target genes were enriched in transcription regulation,protein modification,cell proliferation,apoptosis,differentiation,and kinase activity regulation.The PI3K-Akt signaling pathway,MAPK signaling pathway and TGF-b signaling pathway were significantly enriched.Conclusion: mi R-20a-5p is differentially expressed during lung development and may be involved in the development of RDS.It provides a theoretical basis and experimental basis for its study of lung development-related biological functions and regulatory mechanisms.Part ?: mi R-20a-5p Regulates Pulmonary Surfactant Gene Expression in Alveolar Type II CellsPurpose: Micro RNA(mi RNA)critically controls gene expression in many biological processes,including lung growth and pulmonary surfactant biosynthesis.The present study was conducted to investigate whether mi R-20a-5p had such regulatory functions on alveolar type II(AT-II)cells.Approach: To accomplish this,Mi R-20a-5p overexpression and inhibition adenovirus vector was constructed and transfected into cultured AT-II cells that were isolated from rat fetal lungs of 19 day gestation.Transfection efficiency was confirmed by observing the fluorescence of green fluorescent protein(GFP)carried by the viral vector whereas mi R-20a-5p levels were verified by real time-PCR.The CCK-8 assay was used to compare the proliferation ability of AT-II cells that had over-or under-expressed mi R-20a-5p.The expression of surfactant associated proteins(SPs)and phosphatase and tensin homolog(PTEN)were measured by real time-PCR and Western blotting.Results: In AT-II cells,transfection resulted in over-or under-regulation of mi R-20a-5p.While over-expression of mi R-20a-5p promoted pulmonary surfactant gene expression,its under-expression inhibited it.Consistent with its role in negatively regulating the pulmonary surfactant gene,an opposite pattern was observed for mi R-20a-5p regulation of PTEN.As a result,when mi R-20a-5p was rendered over-expressed,PTEN was down-regulated.By contrast,when mi R-20a-5p was under-expressed,PTEN was up-regulated.Neither over-expression nor under-expression of mi R-20a-5p altered the cell proliferation.Conclusion: mi R-20a-5p plays no role in proliferation of fetal AT-II cells but is a critical regulator of surfactant gene expression.The latter appears to be achieved through a regulatory process that implicates expression of PTEN. |
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