Analysis Of Differentially Expressed MiRNAs In Fetal Lung Tissue Of Sprague - Dawley Rats

With the improvement of treatment rate and livability of the premature infants, the incidence of RDS (Respiratory Distress Syndrome, RDS) and BPD (Bronchopulmonary Dysplasia, BPD) also increases significantly. Now these two diseases have become more and more common in clinical. So, it turns into gradually important to explore the pathogenesis and the new therapeutic targets in the future.The causes of these two diseases are complex. Thereinto, RDS is a common disease in newborns. Nowadays, it has already been widely approved that the lack of alveolar surfactant is an important cause of RDS in the process of lung development. Simultaneously, it has been reported that the occurrence of RDS is also close to gestational age and birth-weight. This may suggest there’s close link between lung development and RDS as well.The essence of BPD has already been widely agreed that an a lung injury lead by various risk factors including oxygentoxicity, mechanical ventilation, infection, inflammation on the basis of genetic susceptibility. Especially in premature infants, because of less alveolars and simpler structure, the whole process of lung development hasn’t been fulfilled yet. Then in the wake of being affected by different risk factors, it will finally lead to lung injury and developmental lag.MiRNAs (microRNAs) is an important kind of short endogenous RNAs found in recent years. They could regulate gene expression in post-transcription level through the direct degradation of the target mRNA or the suppression of their translation. Now, as we know, they play a very important role in many biological processes, such as the cell proliferation, signal transduction, stem cell differentiation, the occurrence and metastasis of the tumor and so on.Biochip technology is anew technology developed since the early1990s, with the characteristics of high throughput, high integration, miniaturization, continuity and the automation.Today this technology is ripely applied in the detection of miRNA expression. So, it is an ideal method and tool to perform miRNA expression profile in cells or tissues.The first part of this paper discusses the morphological research of lung development in fetal rat. The second part discusses the differential miRNA expression of fetal lungs as developing in Sprague-Dawley rats. The third part discusses the possible role of miRNA-126/miRNA-126*in fetal lung development.This experiment uses the miRNA chip technology to screen differentially expressed miRNA spectrum at3time points [Embryo (E)16, Embryo19, Embryo21] of fetal lung development, trying to find meaningful miRNAs close to the lung development. These new found miRNAs may play an important role in lung development, and this result can offers certain physiological basis for neonatal disease on lung development in the future. Part I:Morphological research of lung development in fetal Sprague-Dawley ratsObjective:To study morphological evolution of fetal lungs in Sprague-Dawley rats and establish continuous systematic morphology data.Methods:12healthy pregnant rats have been devided into group S1(E16), group S2(E19), group S3(E21).Then all fetal lungs are isolated at embryonic dayld (E16), E19, E21, respectively. Fetal lungs were subsequently observed by H-E staining (Haemo-toxylin and Eosin staining, H-E) and electric microscope.Results:(1) Under the light microscope:In group S1, epithelial tract began to appear which was constructed by tall epithelium, and developed many airway branches; In grpup S2, alveolar space extended gradually, Then type-Ⅱ pneumonocytes began to emerge; In group S3, alveolar separation increased gradually, mesenchyma turned into thiner.(2) Under the electron microscope:In group S1, there were no lamellar bodys, but chondriosomes; In grpup S2, lamellar bodys began to emerge in type-Ⅱ pneumonocytes; In group S3, lamellar bodys began to increase. The structures of pulmonary epithelium and capillary vessels became clear.Conclusion:The fetal lung development is a gradual process. In fact this process will continue to the postnatal stage. The appearance of lamellar body is the sign of differentiation of the type-II pneumonocytes. and it increases along with lung development. Part Ⅱ:The expression profile of microRNAs in fetal lung development of Sprague-Dawley ratsObjective:Through comparing the miRNA differential expression patterns of3groups (S1, S2, S3), providing the evidence that miRNAs are involved in the molecular pathogensis of fetal lung development.Methods:We used a miRNA microarry to perform the expression profile of miRNAs at3different stages (E16, E19, E21) in the late phase of lung development. After miRNA screening, some miRNAs which have significant changes in miRNA microarray were selected and validated by real-time PCR.Results:(1) In total,167differentially expressed miRNAs were found during fetal lung organogenesis, including81up-regulated miRNAs and86down-regulated miRNAs. As a result of having more than2fold changes in all three groups and showing the tendcy of continuous up-regulation or down-regulation,7miRNAs were selected out for further study.(2) Because of exhibiting higher fold changes, miRNA-125b-5p, miRNA-296, miRNA-93, miRNA-146b and miRNA-3560were then selected for real-time PCR, and the results were basicly coincidence with the tendencies showed in the microarray.Conclusion:These new found miRNAs may play an important role in the process of lung development, including type-Ⅱ pneumonocytes, and may also offer certain physiological basis for researches on lung development diseases in the future. Part Ⅲ:The study of miRNA-126/miRNA-126*’s possible role in fetal lung development of Sprague-Dawley ratsObjective:Through comparing the differential expression patterns of miRNA-126/miRNA-126*in miRNA microarray, to explore the possible roles of these two miRNAs in the process of lung development.Methods:We used a miRNA microarry to perform the expression of miRNA-126/miRNA-126*at3different stages (E16, E19, E21) in rats’lung development. Then, miRNA-126*was selected and validated by real-time PCR because of its significant changes in microarray.Results:After microarray test, miRNA-126/miRNA-126*both showed differential expression between3stages in the fetal lung development. Among them, miRNA-126*was chosen out for further study and validated by real-time PCR because of its higher expression in microarray. And the subsequent result showed the relative expression of miRNA-126*gradually increased between all3groups (Group S1→Group S2→Group S3), which was consistent with the previous microarray result.Conclusion:It’s the first time reported the significant expression of miRNA-126*in fetal lung development, and we guess miRNA-126/miRNA-126*may play an important role in the physiological mechanism of fetal lungs as developing. And this result maybe helpful to the related studies on lung developmental diseases in neonates in the future.