Study On The Mechanism Of MiRNA-26a Affecting The Synthesis Of Lung Surfactants

Respiratory distress syndrome(RDS)is one of the most common critical illnesses in the neonatal period.It has a high morbidity and mortality,and has a serious impact on the quality of life and prognosis of preterm neonates.The key to the development of RDS is due to the relative or absolute deficiency of the pulmonary surfactant(PS).PS is synthesized and secreted by type Ⅱ alveolar epithelial cells(AECⅡ),which can reduce pulmonary surface tension and increase lung compliance to maintain alveolar integrity.The main purpose of this paper is to study the mechanism of neonatal respiratory distress syndrome,to explore the synthesis process of PS,and to provide an important intervention target for disease prevention and drug development.The literatures show that the synthesis and secretion of PS are affected and regulated by many factors.Among them,the role of microRNAs(miRNAs)has been paid more and more attention.Our preliminary studies found that miRNA-26a(miR-26a)is a screened differential expression miRNA in the early stage of pulmonary development.SMAD1,a potential target gene which was closely related to lung development is found by the subsequent bioinformatics analys.SMAD1 is an important signaling molecule in the downstream of the bone morphogenetic protein(BMP)pathway.The key steps in lung embryonic development are almost regulated by BMP.Previous studies have also shown that overexpression of miR-26a can regulate the expression of SMAD1 and inhibit the synthesis of PS in rat fetal lung AECⅡ.Thus,thses suggest that miR-26a plays an important role in lung development and RDS.MiR-26a has little research on the function and mechanism of PS synthesis.In order to explore the signal pathway of miR-26a synthetic PS,first,the pmirGLO-SMAD 1 3’ untranslated region(UTR)double luciferase reporter gene(DLR)expression system can be used to prove that SMAD1 is the target gene of miR-26a;Secondly,the overexpressed and silence SMAD1 lentiviral vectors were constructed,and used to infect A549 cells.The expression levels of SMAD1,PS mRNA were analyzed by real-time PCR.These results found that the expression levels of SP-A and SP·B mRNA were significantly increased after SMAD1 overexpressed,while that was significantly decreased after SMAD1 silence.These results suggest that miR-26a is involved in the regulation of PS by targeting SMAD1.In addition,in order to study the biological function of miR-26a,the homozygous offspring of double-gene knockout mice(miR-26a-1-/-/miR-26a-2-/-)generated by using the CRISPR/Cas9(the clustered regularly interspaced short palindromic repeat/associated protein 9)system were successfully obtained.Then,compared with wild type mice,real-time PCR was performed to detect the expression level of miR-26a in various organs and the mRNA level of PS in the lungs.Moreover,the number of AECⅡ and the synthesis of PS in lung tissue were analyzed by hematoxylin-eosin and immunohistochemical staining.These results showed that the number of AECⅡ cells increased and the synthetic PS increased after miR-26a knockout.This suggest that miR-26a plays an important role in the synthesis of PS in AECⅡ in vivo.Part Ⅰ:miRNA-26a targeting SMAD1 affects the synthesis of PS in A549 cellsObjective:1.To verify that SMAD1 is the target gene of miRNA-26a2.To explore the mechanism of the signal pathway that miRNA-26a targeting SMAD1 affects the synthesis of PSMethods:1.The binding sequence of hsa-miR,26a and SMAD1 3’UTR were obtained in vitro,to recombined with pmirGLO vector plasmid.The pmirGLO-SMAD1 3’UTR double luciferase reporter gene(DLR)expression system was obtained.The different plasmids with hsa-miR-26a-5p mimics were co-transfected into HEK293T cells.Then,the firefly luciferase activity(Ff)and renilla luciferase(Rn)were detected in each treatment group.The relative luciferase activity ratio(Ff/Rn)was calculated and analyzed.2.The construction of overexpression/silence lentivirus and viral empty vector were infected A549 cells,respectively.After 72 hours,total RNA was extracted and real-time PCR was performed to determine the expression level of SMAD1 and PS mRNA.Results:1.The relative fluorescence values of pmirGLO-SMAD1 wt group and hsa-miR-26a-5p mimics co-transfection group were significantly lower than the other two groups,which indicated that miR-26a combined with SMAD1 3’UTR region could reduce the relative fluorescence value Ff/Rn.2.①After 72 hours of infection,up to 95%of the cells had positive expression of green fluorescent protein(GFP).②The expression of SMADI,SP-A and SP-B mRNA was detected by real-time PCR.The results showed that A549 cells were successfully overexpressed/silenced SMAD1;The expression of SP-A and SP-B mRNA in overexpression SMAD1 group was higher than those in control groups in A549 cells,but it in silence SMAD1 group was lower than that in control group.Conclusions:1.SMAD1 is the target gene of miRNA-26a.2.miR-26a can affect PS synthesis by SMAD1.Part Ⅱ:the synthesis of PS in lung of miRNA-26a knockout miceObjective:To study the synthesis of PS in lung of miR-26a knockout mice,and to provide some basis for the reseach of miR-26a on lung development in vivo.Methods:The miR-26a-1-/-/miR-26a-2-/-double knockout mice model was obtained successfully by using the CRISPR/Cas9 system genome editing technique.Compared with wild-type mice,the expression level of miR.26a in various organs and that of pulmonary surfactant protein mRNA in the lungs were detected by real-time PCR in knockout mice.In addition,the number of AECⅡ cells and the expression level of pulmonary surfactant related protein in lung tissue were analyzed by hematoxylin-euphorbia(HE)staining and immunohistochemistry(IHC).Results:1.①The homozygous offspring of miR-26a-1/miR-26a-2 double knockout mice generated by using the CRISPR/Cas9 system were successfully obtained;The sequencing results showed that the deletion fragment was larger than 3bp and miR-26a-1 and miR-26a-2 were knockout simultaneously;②Compared with wild type mice,the expression levels of miR-26a measured by real-time PCR in various organs,in particular the lungs,in the knockout mice were significantly lower.However,the SP-A and SP-B mRNA levels in the lungs of the miR-26a knockout mice were significantly increased.③Compared with wild type mice,HE staining and IHC analysis showed that the number of AECⅡ cells in lung tissue of miR-26a knockout mice were significantly increased,and IHC analysis also found that the expression of pulmonary surfactant-related proteins were also significantly increased in knockout mice.Conclusions:The number of AECⅡ cells and the synthesis of PS were increased after knockout of miR-26a.These suggest that miR-26a plays an important role in the synthesis of PS in AECⅡ cells.