| Objective: BPD is one of the most serious complications of premature infants.The lower the weight and gestational age of premature infants,the higher the incidence.BPD is not only the main cause of early death of premature infants with very low birth weight,but also the main cause of frequent readmission within one year of age.It is also an independent risk factor for undergrowth and long-term nervous system dysplasia,which seriously affects the quality of life of newborns in the future.Therefore,exploring its pathogenesis and seeking effective preventive measures and treatment methods have become one of the urgent problems to be solved.AEC II is the stem cell of alveolar epithelial cells.It is the most vulnerable cell in lung injury.After injury,the proliferation and differentiation process of AEC II appear obstacles,which is an important factor of alveolar dysplasia in lung development.Abnormal transdifferentiation of AEC II,epithelial-mesenchymal transition,is one of the important mechanisms of alveolar dysplasia.It is of great significance to study the mechanism of epithelial-mesenchymal transition of AEC II and how to inhibit or reverse the transition of epithelial-mesenchymal.BMP7 is one of the members of transforming growth factor-beta superfamily.It has a wide range of biological functions and can regulate the differentiation,proliferation and apoptosis of many kinds of cells.Studies have shown that BMP7 can inhibit and reverse the transdifferentiation of epithelial cells into mesenchymal cells.In recent years,BMP7 has been found to play an important role in the pathophysiological process of lung tissue.In my previous study,we found that BMP7 was differentially expressed in the lung tissues of BPD newborn rats.In order to confirm whether the abnormal expression of BMP7 exists in AEC II of BPD and whether the abnormal expression of BMP7 activates the downstream signal transduction protein smad,we detected BMP7 and its signal transduction protein Smad1 and smad2/3 in the AEC II of the BPD model group and the control group,and confirmed that BMP7 may regulate EMT of alveolar epithelial cells by regulating the phosphorylation of smad2/3 protein to participate in the occurrence and development of BPD.we used rat lung epithelial cell line RLE-6TN of ATCC to intervene with exogenous BMP7 to observe the changes of cell biological function,transdifferentiation index,BMP7 and downstream transduction proteins smad1 and smad2/3.In order to regulate the expression of BMP7 to avoid or reduce the occurrence of EMT,combined with the previous study of MicoRNA screening of BPD model rat specimens by gene chip technology.We screened out differentially expressed micoRNA,and verified by enlarging sample size,and predicted whether differentially expressed micoRNA could target BMP7 by bioinformatics.On the basis of confirming that the expression abundance of BMP7 in BPD decreased gradually,and that the expression abundance of microRNA-365-3p in BPD increased gradually,we applied rat alveolar epithelial cell line and used double fluorescence technology to prove the binding relationship between them,used gene transfection technology to observe the changes of EMT-related markers,BMP7 and downstream signal transduction protein smad,and identified the process of regulating epithelial-mesenchymal transformation by down-regulating BMP7 expression targeting by miR-365-3p.Provide evidence for the use of miR-365-3p as a specific target for future BPD therapy.Methods: The BPD model of newborn rats was established according to our previous methods.200 newborn SD rats were randomly divided into BPD group and control group.The newborn rats in BPD group inhaled 80% oxygen,while the newborn rats in control group inhaled air.On 3,7 and 14 days after the experiment,10 newborn rats were randomly selected to separate lung tissue.Lung histomorphology was observed by HE staining.Primary AEC II was extracted,purified,cultured and identified according to our previous methods.The expression of BMP7,Smad1 and smad2/3 protein in purified AEC II cells was localized by immunofluorescence.The expression of BMP7,smad2/3 and p-smad2/3 protein in AEC II cells was determined by Western blot.The expression of BMP7,smad1,smad2 and smad3 mRNA in AEC II cells was determined by qRT-PCR.Cryopreserved RLE-6TN cells were recovered,cultured and passaged from liquid nitrogen.The well-growing cells were digested and inoculated into 6-well plates.The cells were divided into four groups according to different treatment factors: control group: normal RLE-6TN monolayer cells.BMP7 group: RLE-6TN monolayer cells were treated with 10ng/ml BMP7.EGF group: RLE-6TN monolayer cells were treated with 20 g/ml EGF.BMP7 + EGF group: 10ng/ml BMP7 and 20ng/ml EGF were added to the RLE-6TN monolayer.Cells were collected for 48 hours.The morphological changes of RLE-6TN cells were observed under light microscopy,the horizontal migration of cells was observed by cell scratch test,the expression of E-cad/N-cad,Spb/vimentin in RLE-6TN cells was localized by double immunofluorescence staining,the expression of signal transduction proteins smad1 and smad2/3 in RLE-6TN cells was localized by immunocytochemistry staining,and the expression of E-cad,N-cad,vimentin,smad1,p-smad1,smad2/3 and p-smad2/3 in RLE-6TN cells was determined by Western blot.The expression of E-cad,N-cad,vimentin,smad1,smad2 and smad3 in RLE-6TN cells were detected by qRT-PCR.The differentially expressed micoRNA was screened.The expression of miR-365-3p in BPD lung tissue and AEC II was determined by microRNAs qRT-PCR.RLE-6TN cells overexpressed miR-365-3p,and the transfection efficiency was detected by microRNAs qRT-PCR.The morphological changes of RLE-6NT cells transfected with miR-365-3p mimics were observed under light microscope.Western blot was used to detect the expression of N-cad and E-cad after miR-365-3p mimics transfection.The expression of interstitial marker N-cad and epithelial marker E-cad was detected by qRT-PCR after transfection of miR-365-3p mimics.Western blot was used to detect the expression of BMP7,smad2/3 and p-smad2/3 protein after miR-365-3p mimics transfection.The expression of BMP7,smad2 and smad 3 was detected by qRT-PCR after miR-365-3p mimics transfection.Double luciferase reporter gene was used to verify the binding of miR-365-3p to BMP7.Results: 1.Histological observation of lung showed that with the prolongation of time,the number of alveoli decreased,secondary septation decreased and RAC value decreased in BPD group,suggesting that alveolar development was stagnated.Immunofluorescence results showed that BMP7 expression in BPD group was significantly higher than that in control group,both cytoplasm and nucleus were visible.The fluorescence intensity of BMP7 gradually decreased with the increase of age,and the red fluorescence of BMP7 could not be seen in the nucleus of 14 d BPD group.BMP7 was expressed in the control group,and the expression was mainly localized in the cytoplasm.Western blot showed that the expression of BMP7 protein in BPD group was higher than that in control group on the 3rd and 7th day of experiment,and the expression of BMP7 protein in BPD group decreased gradually with the increase of age.qRT-PCR showed that BMP7 gene expression in BPD group was significantly higher than that in control group,but decreased with age.2.Immunofluorescence results showed that the green fluorescence of smad1 was mainly localized in the cytoplasm,but no expression of green fluorescence was found in the nucleus.Smd2/3 expression in BPD group was higher than that in control group.Green fluorescence was mainly localized in cytoplasm and nucleus.In BPD group,smad 2/3 increased most significantly at 7 days,and the green fluorescence was localized in the nucleus,with only a small amount of green fluorescence in the cytoplasm.The expression of smad2/3 decreased in BPD group at 14 days,and the green fluorescence was mainly concentrated in the cytoplasm.The fluorescence intensity was slightly lower than that of the control group.The expression of smad2/3 in BPD group increased first and then decreased.Western blot showed that the expression of p-smad2/3 protein increased and then decreased with age in BPD group.The total protein expression of smad2/3 decreased gradually.qRT-PCR showed that the expression of Smad1 in BPD group decreased first and then increased.The expression of smad2 in BPD group was significantly higher than that in control group.The expression of smad2 in BPD group increased with age.The change trend of smad3 expression was the same as that in smad2.3.Under inverted phase contrast microscope,BMP7 cells still maintained epithelioid morphology,and the cell-to-cell junction was close.In EGF group,the cells were loosely connected,the gap became larger and filamentous pseudopods were formed,showing fibroblast-like appearance.In BMP7+EGF group,the intercellular junction was tighter than that in EGF group,and the filopodia was less.BMP7 group did not affect cell migration ability,EGF group could increase cell migration ability,BMP7+EGF group decreased cell migration ability,inhibited cell migration to a certain extent.Immunofluorescence double staining showed that the expression of E-cad in EGF group was weaker and that of N-cad was stronger.The expression of E-cad in BMP7+EGF group increased significantly and that of N-cad decreased significantly.Spb expression was weaker in EGF group and Vimentin expression was stronger;Spb increased significantly in BMP7+EGF group and Vimentin decreased significantly.Western blot showed that the expression of N-cad and Vimentin in control group and BMP7 group was lower than that in EGF group,while that in BMP7+EGF group was lower than that in EGF group,but higher than that in control group and BMP7 group.The expression of E-cad in control group and BMP7 group was higher than that in EGF group.The expression of E-cad in BMP7+EGF group was higher than that in EGF group,but lower than that in control group and BMP7 group.The expression of N-cad and Vimentin in control group and BMP7 group was lower than that in EGF group,while that in BMP7+EGF group was lower than that in EGF group.The expression of E-cad in control group and BMP7 group was higher than that in EGF group.The expression of E-cad in BMP7+EGF group was higher than that in EGF group,but lower than that in control group and BMP7 group.4.Immunocytochemical staining showed that small amount of smad1 protein was expressed in control group,but not in BMP7 group,EGF group and BMP7+EGF group.Smad1 protein was mainly localized in the cytoplasm,and only small amount of smad1 expression was found in the nucleus.Immunocytochemical staining showed that a large number of smad2/3 proteins were localized in the nucleus of BMP7 and BMP7+EGF groups,and brown granules were seen in the nucleus and cytoplasm.Smd2/3 protein was expressed in control group and EGF group,and smad2/3 protein was mainly expressed in cytoplasm.Western blot showed that the expression of smad1 protein in BMP7,EGF and BMP7+EGF groups was lower than that in control group.Compared with control group,the expression of p-smad1 protein increased slightly in BMP7 group,but decreased in EGF group and BMP7+EGF group.The expression of smad2/3 protein in control group and BMP7 group was basically the same.The expression of smad2/3 protein in EGF group was significantly lower than that in control group,and the expression of smad2/3 protein in BMP7+EGF group was higher than that in EGF group.The expression of p-smad2/3 protein in BMP7 group was significantly higher than that in control group.The expression of p-smad2/3 protein in EGF group and BMP7+EGF group was higher than that in control group.qRT-PCR showed that the expression of smad1 mRNA in each treatment group was lower than that in control group.Compared with control group,the expression of smad2 mRNA in BMP7 group was significantly increased.There was no difference in the expression of smad2 mRNA between EGF group and control group.The expression of smad2 mRNA in BMP7 group was significantly higher than that in EGF group.The expression of smad2 in BMP7+EGF group was higher than that in control group,but lower than that in BMP7 group.The change trend of smad3 mRNA expression was basically the same as that of smad2 mRNA.5.Through TargetScan Biological Information Prediction Website,combined with the results of gene chip detection,24 differentially expressed micoRNAs were screened out,of which the binding sites of miR-365-3p and BMP7 may exist.The expression of microRNAs qRT-PCR in lung tissue and AEC II was higher in BPD group than in control group.The efficiency of over-expression of miR-365-3p was verified by microRNAs qRT-PCR,and the efficiency of over-expression was significant.Cell morphology observation showed that the cells in control group and NC group were epithelioid cells with obvious polarity,tight junction between cells and cells,loose junction between cells in miR-365-3p group,large gap and obvious filamentous pseudopod formation,showing fibroblast-like changes.Western blot showed that the expression of N-cad protein increased and E-cad protein decreased after miR-365-3p mimics transfection.qRT-PCR showed that the expression of N-cad increased and E-cad decreased after the transfection of miR-365-3p mimics.Western blot showed that the expression of BMP7,p-smad2/3 and smad2/3 protein in transfected miR-365-3p mimics was lower than that in control group.qRT-PCR showed that the expression of BMP7,smad2 and smad3 in miR-365-3p mimics transfected group was lower than that in control group and NC group.The results of double luciferase reporter gene assay showed that co-transfection of BMP7-wt and miR-365-3p mimics could significantly decrease the luciferase activity of 293 T cells.miR-365-3p could bind to BMP7.Conclusion: 1.Previous studies have confirmed that in the lung tissue of BPD,BMP7 expression was increased,and extended gradually decreases over time.This study found that in type ? alveolar epithelial cells,BMP7 expression change time synchronization with organization,and its downstream signal transduction protein smad2/3 transfer activation which indicates that the pathway play a role in the occurrence of BPD.2.Vitro experiments confirmed that after EGF induced lung epithelial cells,BMP7 inhibits alveolar epithelial differentiation by activating signal transduction protein smad2/3,the finding offers a new approach for clinical application.3.The high expression of miR-365-3p in BPD was verified at tissue and cell levels respectively.MiR-365-3p can target BMP7,down-regulate BMP7 and reduce the activation of downstream signal transduction protein smad2/3,which is an important mechanism of pulmonary epithelial cells to mesenchymal differentiation and provides a new way for the prevention and treatment of BPD in the future. |
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