| Neonatal respiratory distress syndrome(RDS)is one of the serious diseases of newborns with high morbidity and mortality and is the leading cause of children’s chronic lung disease. Although preventive steroid and exogenous pulmonary surfactant(PS) has been applied, the clinical efficacy of RDS is not just as good as expected. Therefore, the prevention and treatment of RDS have always been a hot research subject in the newborn over the years.RDS is caused by interaction of gene mutation, serious infection, hypoxia and other internal and external factors, which have been proved by clinical medicine, epidemiology and biochemistry in recent years. It may either directly damage the alveolar type II cells or damage alveolar capillary endothelial cells by inflammatory reaction and subsequently lead to increased vascular permeability, plasma and fibrinogen exudation, causing the formation of alveolar hyaline membrane, leading to primary or secondary insufficient SPs secretion. However, the mechanism of many factors on this disease are unclear so far. The basic way of preventing RDS is to further explore its pathogenesis.The latest research suggests that micro RNAs(mi RNAs) may be involved in the regulation of all the cellular mechanism, the expression of mi RNA is changed in many diseases and mi RNA has been involved in the pathogenesis of these diseases. At present, research on mi RNAs mainly focuses on the field of cancer, but the research about mi RNA in the field of RDS is rare.Our group found that mi R-26 a is differentially expressed and gradually upregulated at the different late stages of fetal lung development by micro RNAs microarray analysis. Besides, blood micro RNAs microarray in RDS group and the normal group showed that the two groups had a significant difference in mi R-26 a expression, the expression of mi R-26 a in RDS group was significantly higher than that in normal group. Mi R-26 a is highly conserved in vertebrates and has been reported to play an important role in tumorigenesis, organ development, cell proliferation, and other biological processes. Therefore, we speculated that mi R-26 a might have a certain impact on fetal lung development and respiratory distress syndrome.This project was divided into two parts, the first part was to predict the target genes of mi R-26 a, and to analyse its application in lung development. The second part was to explore the regulation role of mi R-26 a in the synthesis of pulmonary surfactant.This study discusses the effect of mi R-26 a on the synthesis of pulmonary surfactant of fetal rat type II alveolar epithelial cells, which may provide new research targets and theoretical clues for further studies of the pathogenesis of RDS.Part I:Prediction of mi R-26 a Target Genes and Bioinformatics AnalysisObjective: The present study aimed to bioinformatically analyze as well as predict the target genes of mi R-26 a, so as to lay foundation and provide theoretical basis for the further studies of biological function of mi R-26 a in fetal lung development.Methods: All literature of mi R-26 a was searched in pubmed and Google; mi RBase database was used to obtain the sequence of mi R-26 a and to analysize its conservation. The target genes of mi R-26 a was predicted using mi RNA target gene database mi RGen. Target Scan and Pic Tar were used to predict target genes of hsa-mi R-26 a. The intersection of the two results and validated targets from DIANA LAB-Tar Base6.0 datebase was analyzed by gene ontology and pathway analysis.Results: Mi R-26 a had been reported in cancer, cell differentiation, organ development, the immune system as well as involved in other biological process; mi R-26 a was highly conservative among different species. Target genes exist in all of the cell components, including cell membrane, cytoplasm and nucleus.The functions of these target genes were enriched in translation regulation, the protein modification, the regulation of celluar proliferation, apoptosis and differentiation process, kinase activity regulation. The Wnt signaling pathway, MAPK(mitogen-activated protein kinase) signaling pathway, TGF-beta(transforming growth factor beta), the pathway of tumor p53 signaling pathways, cell cycle and adherens junction pathways were significantly enriched.Conclusions: Mi R-26 a may play an important role in lung development and some physiological and pathological processes such as neonatal respiratory distress syndrome through BMP signal pathway, Wnt pathway, TGF-beta pathway and so on.PartⅡ:Regulation of Pulmonary Surfactant Synthesis in Fetal Rat Type II Alveolar Epithelial Cells by mi R-26aObjective: To explore the regulation role of miR-26 a in the synthesis of pulmonary surfactant.Methods: Mi R-26 a genomic sequence was amplified by polymerase chain reaction(PCR) and cloned into GV264 plasmid, the recombinant plasmid was generated in colon bacillus. Then, the recombinant plasmid was co-transformed into human embryonic kidney293(HEK293)cells with an adenoviral backbone plasmid p BHG lox ΔE1,3 Cre. The linearized Ad-26 a was packed and amplied in HEK293;Lung tissues of 19-day fetal rats were digested with trypsin and collagenase, then screened for AECII with different centrifugal force and repeated attachment. Growth status and shape of attached cells were observed with inverted phasecontrast microscope. AECII were identified by electron microscopy and its percentage was assessed by detecting the expression of surfaetant protein C(SP-C) in AECII by immunofluorescence; Ad-26 a was introduced into primary cultured fetal AECII. The expression of green fluorescent protein in alveolar epithelial type II cells was observed by fluorescence microscopy. The expression of mature mi R-26 a was detected by real time PCR; MTT was performed to analyze AECII viability; q RT-PCR and western blotting were used to determine the m RNA and protein level of SMAD1 and surfactant-associated proteins.Results: 48 h after transfection, expression of green fluorescent protein was detected in 93% AECII under a fluorescence microscope, real-time PCR showed that mi R-26 a was up-regulated significantly in AECII infected with Ad-26a(P<0.05), the result showed that theadenovirus vector Ad-26 a was constructed successfully and canexpress efficiently in AECII; the overexpression of mi R-26 a inhibited the synthesis of SP-B and SP-C in m RNA level and inhibited the synthesis of SP-Cinprotein level in AECII(P<0.05). There was no significant change in cell proliferation(P>0.05). Our results further showed that overexpression of mi R-26 a reduced the SMAD1 expression both in m RNA and protein level in fetal AECII(P<0.05).Conclusion: These findings indicate that mi R-26 a regulates surfactant synthesis in fetal AECII through SMAD1, which may provide new research targets and theoretical clues for further study of pathogenesis of RDS.
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