Pathogenesis Of Pulmonary Dysplasia Through MiR-455-5p Regulate STRA6 In Nitrofen-induced Congenital Diaphragmatic Hernia

ObjectivesThe goal of this study was to investigate the pathogenesis of pulmonary dysplasia through miR-455-5p regulate STRA6 in Nitrofen-induced congenital diaphragmatic hernia,to clarify the relationship between miR-455-5p-STRA6 ?pulmonary dysplasia of CDH,to explore the etiology of CDH and to provide theoretical basis and experimental basis for the prevention of pulmonary dysplasia of CDH and explore new prenatal intervention methods.Methods1.The upstream mircroRNAs of STRA6 gene were predicted by bioinformatics software2.Dual luciferase reporter genetechnology to verify the regulation relationship between miR-455-5p and STRA6.3.To set up Nitrofen-induced the rats model of pulmonary dysplasia of congenital diaphragmatic hernia.And then,the expression of miR-455-5p and STRA6 mRNA in the lungs of 15-day-old fetal rats were examined by real-time quantitative polymerase chain reaction through this rat model.Beside the changes of vitamin A density were detected by ELISA.4.The relative expession level of STRA6 protein in the CDH model were detected by using Immunohistochemistry and immunohistochemistry.5.The miR-455-5p interfering adenovirus and overexpressing adenovirus were set up;Rat lung epithelial cell lines were cultured and passaged,virus transfection after 72 hours,the expression of STRA6 proteinwere detected by Western Blotting,the changes of vitamin A density in fetal lung were detected by ELISA and cell proliferation were detected by CCK8 after the change of miR-455-5p level.Result1.The results of the upstream mircroRNAs of STRA6 gene were predicted by bioinformatics softwareThree software co-predictions indicate that the mircroRNA upstream of the STRA6 gene is miR-455-5p.2.The results of Dual luciferaseThe average relative expression of fluorescence values of STRA6-WT + NC,STRA6-WT + no-miR-455-5p,STRA6-Mut + NC,STRA6-Mut + no-miR-455-5p were 1.000 ± 0.023,0.521 ± 0.025,1.000 ± 0.014 and 0.768 ±0.008,respectively.Rno-miR-455-5p mimic showed a significant down-regulation of the reporter fluorescence in STRA6 wild type(47.9%).After mutagenesis of its predicted target site,the reporter fluorescence in the mutant vector were recoveredpartially,but the result is still significant(23.2%).miR-455-5p can regulate the expression of STRA6 gene.3.The results of animal model,miR-455-5p,STRA6 mRNA expression were detected by QPCR and the changes of vitamin A density were detected by ELISA.The outcome of Nitrofen-induced rat model of pulmonary dysplasia of CDH:In the control group,62 fetal samples were obtained by cesarean section in 6 pregnant SD rats.In Nitrofen-induced group,45 fetal samples were obtained by cesarean section in 6 pregnant SD rats.The were fetal samples were distributed in various experimental methods equally.On D15.5,the relative expression of miR-455-5p in the control group and Nitrofen-induced group were 5.955±2.330 and 29.450±9.253 respectively.the relative expression of miR-455-5p in Nitrofen-induced group was increased significantly by comparing to the control group(P<0.05);The relative expression of STRA6 mRNA were 0.588±0.184 in the control group and were 0.197±0.097 in Nitrofen-induced group.On the contrary,the relative expression of STRA6 mRNA in Nitrofen-induced group was lower significantly than those in the control group(P<0.05);The average density of vitamin A in the Nitrofen group and the control group was 4.03 ±0.195 and 5.317 ± 0.337,and the average density of vitamin A in the Nitrofen group was significantly lower than that in the control group(P = 0.000<0.05).4.The results of Immunohistochemistry and Immunofluorescence histochemistryImmunohistochemistry:The average H-SCORE values of STRA6 protein in the control group were 156.242 ± 9.922,at the same time,the Nitrofen-induced group were 120.020 ±8.514.By contrast to the control group,the relative expression of STRA6 protein was significantly cut back in the Nitrofen-induced group,which were significant difference between then(P<0.05).Immunofluorescence histochemistry:The average IOD values of STRA6 protein in the control group and Nitrofen-induced group were 1616.391 ± 572.308 and 805.643± 291.182 respectively.After statistical analysis,significant differences were observed in the two groups.(P<0.05).The relative expression of STRA6 protein was lessened obviously in the Nitrofen-induced group.5.The results of miR-455-5p overexpression influences the regulation of STRA6?the metabolism of vitamin A and the proliferation of cells in the rat lung epithelial cellThe results of Western Blotting were showed that the relative expression levels of STRA6 protein in NC group were 0.958 ± 0.246,and in miR-455-5p overexpression group were 0.615 ± 0.131 which was manifest decrease in comparison with the NC group(P<0.05).The results of ELISA were displayed that the average density in miR-455-5p overexpression group and NC group were 2.985±0.061,3.949±0.118,respectively.The density of vitamin A in miR-455-5p overexpression group was diminished apparently through compared with the NC group(P<0.05).CCK8 experimental results:at 24h,48h,72h,96h,miR-455-5p overexpression group and NC group,the average absorbance were 0.280 ± 0.184 and 1.354 ± 0.169,0.881 ± 0.016,and 1.992 ± 0.050,2:937±0.079 and 2.105 ± 0.148,4.955±0.0021 and 3.315 ± 0.043,respectively.At 4 time points,the average absorbance of miR-455-5p overexpression group was significantly lower than NC groups(P<0.05).Compared with the same group,the average absorbance value of the two groups showed an increasing trend,which was statistically significantat difference(P<0.05).ConclusionIn the Nitrofen-induced model of pulmonary dysplasia of congenital diaphragmatic hernia,the abnormal increase of miR-455-5p resulted in partial degradation of the STRA6 mRNA of the target gene,resulting in a decrease in the target STRA6 protein,so that Vitamin A transport disorder and the density of vitamin A were decreased in fetal rat lung.In rat lung epithelial cells,miR-455-5p overexpression can down-regulate the expression of STRA6 gene,so that cell metabolism of vitamin A was abnormalities,the density of vitamin A went down,Vitamin A transport disorder and then cell proliferation were decreased.Therefore,combined with the experimental results above,we think miR-455-5p abnormal level is one of the reasons of congenital diaphragmatic hernia lung dysplasia.