Screening Of Differential Expression Profiles Of Long Non-coding Rna In Childhood Asthma And Its Function And Mechanism Study

Background:Bronchial asthma(asthma)is one of the most common chronic respiratory diseases in the world.According to statistics,there are about 334 million asthma patients in the world by 2015^[1].In recent years,the prevalence rate of asthma in children is increasing year by year.The third epidemiological survey of asthma in urban children in China shows that the prevalence rate of asthma in urban children in China has increased to 3.02%from 1.97%ten years ago.The prevalence rate of asthma in children has increased by 54.1%^[2].It is worth noting that in China,about one percent children with asthma have not been timely and clearly diagnosed,and nearly half of the children's asthma has not been effectively controlled,resulting in persistent damage of lung function^[3].Therefore,it is of great significance to clarify the mechanism of childhood asthma and to find the target for the diagnosis and treatment of childhood asthma.At present,it is generally believed that airway epithelial cells can participate in the occurrence of airway remodeling in asthma in the form of epithelial-mesenchymal transformation(epithelialmesenchymaltransition,EMT)under the stimulation of environmental factors.How to regulate the EMT process of bronchial epithelial cells so as to reduce airway remodeling has become the focus of prevention and treatment of asthma.Long non-coding RNA(longnon-coding RNA,lncRNA)is a class of RNA molecules with a length of more than 200bp.It exists widely in eukaryotes and participates in the regulation of various processes in cells,but it can not encode proteins.^[4].Compared with protein coding m RNA,their expression abundance is generally lower,but they have highly conservative secondary structure and stronger tissue cell type specificity^[5].Because of this characteristic,lncRNA has become a potential molecular marker for the diagnosis and prognosis of many diseases,and the study of its functional mechanism provides a target for the treatment of diseases.At present,some studies have sequenced the whole blood samples or peripheral blood T lymphocytes of adult asthma patients,and found that there is differential expression of lncRNA in asthma patients,suggesting that lncRNA may be involved in the regulation of immune,airway inflammation and other pathological processes of adult asthma from the perspective of epigenetics^[6-10].However,studies on the function and clinical significance of most asthma-related lncRNA are still limited.Therefore,analyzing the differential expression profile of lncRNA in children with bronchial asthma and looking for the lncRNA,which may be related to the pathogenesis and disease progression of childhood bronchial asthma may provide a new direction and therapeutic target for the prevention and treatment of bronchial asthma,slow down the process of airway remodeling and prevent the progressive aggravation of pulmonary function damage.Objective:To establish the differentially expressed lncRNAs and m RNAs expression profiles in peripheral blood of children with asthma,and to obtain the molecular function,biological process and enriched disease pathway information of differentially expressed m RNAs.At the same time,we screened and verified the biological function and potential regulatory mechanism of target lncRNA in the process of bronchial epithelial cell EMT,so as to provide theoretical basis and therapeutic targets for further exploring the pathogenesis,prevention and treatment of asthma in children.Methods:1.The differential expression profiles of lncRNAs in peripheral blood of children with asthma were obtained by lncRNA/m RNA gene chip.The target lncRNA,was screened by bioinformatics analysis and the expression changes of target lncRNA and its associated mi RNA and m RNA in peripheral blood of children with asthma were verified.1)5ml venous blood was collected from all subjects,and PBMCs was isolated byFicoll density gradient centrifugation.2)The total RNA,of the sample was extracted by Trizol method and the qualitywas tested.3)6 PBMCs samples(3 cases in asthma group and 3 cases in healthy controlgroup)were selected and analyzed by Arraystar Human LncRNA Microarray4.0chip.4)The differentially expressed LncRNA or differentially expressed m RNAbetween the two groups were screened by P-value/FDR.The differentially expressed LncRNAs or differentially expressed m RNAs between the two samples were screened by Fold Change.Differentially expressed m RNAs was used for enrichment analysis of Pathway and GO,and scripts were used for hierarchical clustering and association analysis.5)The PPI network of differentially expressed genes was constructed bybioinformatics method,and hub genes were further screened.Establish the network map of the association between lncRNA and hub genes,and search for the related gene pairs between hub gene and mi RNA through mi RWalk database.Finally,the mi RNA-m RNA gene pairs and lncRNA-mi RNA gene pairs are intersected,and the lncRNA-mi RNA-m RNA gene pairs are obtained,and the target lncRNA is screened out.6)Quantitative reverse transcription polymerase chain reaction(q RT-PCR)wasused to verify the expression of target lncRNA and its associated mi RNA and m RNA in a large sample of PBMCs.2.The effect of lncRNA PTTG3P on EMT and its proliferation and migration ability of bronchial epithelial cells.1)The first part of this study finally screened and determined that lncRNAPTTG3P may play an important role in childhood asthma.Bronchial epithelial cell line(16HBE)was cultured in vitro and the cell model was established.16HBE cells were stimulated by TGF-?1,and the morphology of cells was observed under microscope.The expression of EMT markers such as E-cadherin protein,Vimentin protein and?-SMA protein was detected by Westen Blot.To determine the optimal concentration and time of EMT induced by TGF-?1 in 16HBE.2)To determine the expression of lncRNA PTTG3P in bronchial epithelial cellsinduced by TGF-?1 in the state of EMT.q RT-PCR was used to detect the expression of lncRNA PTTG3P.3)The changes of EMT markers such as E-cadherin protein,Vimentin protein and?-SMA protein were detected by small interference RNA(smallinterference RNA,si RNA)knock-down PTTG3P,Westen Blot technique,and the morphological changes of cells were observed.At the same time,CCK-8 test and transwell test were used to detect the proliferation and migration ability of bronchial epithelial cells,and to determine the effect of knocking down lncRNAPTTG3P on the EMT process,proliferation and migration of bronchial epithelial cells.3.Study on the mechanism of lncRNA PTTG3P promoting EMT in bronchial epithelial cells1)The first part of this study finally screened and determined that lncRNAPTTG3P may play an important role in childhood asthma.At the same time,combined with bioinformatics analysis and large sample verification of q RT-PCR,it was found that mi R-192-3p and CCNB1 were also differentially expressed in childhood asthma,which may become downstream factors regulated by lncRNA PTTG3P.So in the in vitro model of bronchial epithelial cell EMT induced by TGF-?1,q RT-PCR and Westen Blot methods were used to detect its expression level and to determine its expression trend.2)The expression levels of mi R-192-3p and CCNB1 were detected by si RNAknockdown PTTG3P,q RT-PCR and Western Blot methods,respectively,todetermine the effect of PTTG3P on the expression of mi R-192-3p and CCNB1.3)The changes of E-cadherin protein,?-SMA protein and Vimentin protein weredetected by overexpression of mi R-192-3pcent WB,and the morphological changes of bronchial epithelial cells were observed to clarify the regulation of mi R-192-3p on EMT of bronchial epithelial cells.4)The regulatory effect of mi R-192-3p on CCNB1 was confirmed byoverexpression of mi R-192-3p,Westen Blot and q RT-PCR to detect the expression level of CCNB1.5)The direct binding of mi R-192-3p to lncRNA PTTG3P and CCNB1 was provedby double luciferase reporter gene assay.6)Through the rescue experiment of knocking down PTTG3P and low expressionof mi R-192-3p,it is proved that the effect of knocking low PTTG3P on promoting EMT in bronchial epithelial cells can be antagonized by low expression of mi R-192-3p.It is proved that PTTG3P may up-regulate CCNB1and promote the action mechanism of EMT in bronchial epithelial cells by adsorbing mi R-192-3p.Results:1.There is a specific lncRNAs expression profile in the peripheral blood of children with asthma.LncRNA PTTG3P is highly expressed in the peripheral blood of children with asthma,mi R-192-3p is low in the peripheral blood of children with asthma,and CCNB1is highly expressed in the peripheral blood of children with asthma.1)A total of 397 differentially expressed 1nc RNAs were screened,of which193were up-regulated and 204were down-regulated.LncRNAs expression was up-regulated and lncRNAs expression was down-regulated.A total of 477differentially expressed m RNAs were screened,of which 398 up-regulated m RNAs expression and 79 down-regulated m RNAs expression.2)GO analysis of differentially expressed genes showed that DEm RNAs wasenriched in"cell cycle"and"binding to protein",while Pathway analysis showed that DEm RNAs was enriched in cell cycle,immune regulation,p53 and other related signal pathways.3)Gene chip and q RT-PCR double proved that the expression of PTTG3P inPBMCs of children with asthma was significantly increased.Through bioinformatics analysis,it was further found that mi R-192-3p and CCNB1 may be associated with PTTG3P.4)QRT-PCR showed that the expression of mi R-192-3p was down-regulated inasthma group,while the expression of CCNB1 was up-regulated in asthma group,the difference was significant.2.In the 16HBE cell model stimulated by TGF-?1 in vitro,knocking down lncRNA PTTG3P can inhibit the change of EMT and inhibit the proliferation and migration of16HBE cells.1)After 16HBE cells were treated with different concentrations of TGF-?1 for 48hours,the expression level of E-cadherin protein decreased gradually with the increase of TGF-?1 concentration(p<0.01),Vimentin and?-SMA protein expression increased gradually).After 16HBE cells were treated with 10ng/ml TGF-?1 for different time,the expression level of E-cadherin protein in 16HBE cells decreased gradually with the prolongation of TGF-?1 treatment time(p<0.01),Vimentin and?-SMA protein expression level gradually increased),thus the best concentration of TGF-?1 was 10ng/ml and the best treatment time was48 hours.After treated with 10ng/ml TGF-?1 for 24 hours,the morphology of16HBE cells changed from round or oval paving stone to long fusiform,and EMT changed.2)After 48 hours of treatment with 10ng/ml TGF-?1,the expression of PTTG3Pin 16HBE cells was significantly up-regulated.3)The si-PTTG3P,with the highest knock efficiency was screened out by si RNAtechnique,which could significantly down-regulate the expression of lncRNA PTTG3P(p<0.005).Knockdown of PTTG3P could up-regulate the expression of epithelial marker E-cadherin protein and down-regulate the expression of stromal cell marker Vimentin and?-SMA protein(p<0.05).In addition,knocking down PTTG3P also helps to maintain cell epithelioid morphology.4)CCK-8 assay and transwell results showed that knocking down PTTG3P couldinhibit the proliferation and migration of 16HBE cells.3.In the EMT model of 16HBE cells induced by TGF-?1 in vitro,the overexpression of mi R-192-3p down-regulated the expression of CCNB1 and inhibited the process of EMT,while the low expression of mi R-192-3p could antagonize the inhibitory effect of lncRNA PTTG3P on EMT and the expression of CCNB1 protein.1)In the EMT model of 16HBE cells induced by TGF-?1,the expression ofmi R-192-3p decreased and the expression of CCNB1 increased with time.2)In the 16HBE cell model induced by TGF-?1,knockdown of PTTG3P coulddown-regulate the expression of CCNB1 protein and m RNA,and up-regulate the expression of mi R-192-3p.3)In the 16HBE cell model induced by TGF-?1,overexpression of mi R-192-3pcould up-regulate the expression of epithelial marker E-cadherin and down-regulate the expression of interstitial marker Vimentin and?-SMA.Overexpression of mi R-192-3p also contributes to the maintenance of epithelioid morphology of 16HBE cells.4)Overexpression of mi R-192-3p could down-regulate the expression of CCNB1protein and m RNA in 16HBE cells induced by TGF-?1.5)The results of double luciferase kit showed that mi R-192-3p could significantly down-regulate the luciferase activity of p SI-check2-PTTG3P and p SI-check2-CCNB1-3'UTR(p<0.05),but there was no difference in luciferase activity between p SI-check2-PTTG3P-mut and p SI-check2-CCNB1-3'UTR-mut groups.It is suggested that mi R-192-3p can be combined with PTTG3P and CCNB1 3'UTR.6)Compared with the group transfected with si-PTTG3P alone,the expression ofE-caherin protein decreased in si-PTTG3P and mi R-192-3p inhibitor group(p<0.05).The expression of),Vimentin,?-SMA and CCNB1 protein increased(p<0.05).It is suggested that mi R-192-3p can antagonize the effect of knockdown PTTG3P on EMT process and CCNB1 protein induced by TGF-?1 in 16HBE cells,indicating that PTTG3P can induce EMT in 16HBE cells induced by TGF-?1.It may be achieved through the regulation of mi R-192-3p and CCNB1.Conclusions:1.LncRNA/m RNA gene chip screening showed that lncRNA PTTG3P was highly expressed in the peripheral blood of children with asthma,and lncRNA PTTG3P-related CCNB1 m RNA was also highly expressed in the peripheral blood of children with asthma.2.Based on the bioinformatics analysis of lncRNA PTTG3P and CCNB1 m RNA,it was found that the expression of mi R-192-3p was low in the peripheral blood of children with asthma.3.EMT changes can occur in bronchial epithelial cells induced by TGF-?1,and the expression of lncRNA PTTG3P in bronchial epithelial cells induced by TGF-?1 is up-regulated.4.LncRNA PTTG3P can promote the EMT process of bronchial epithelial cells induced by TGF-?1,and enhance the proliferation and migration of bronchial epithelial cells.5.When TGF-?1 induced EMT in bronchial epithelial cells,the expression of mi R-192-3p was down-regulated,and mi R-192-3p could specifically regulate the inhibition of CCNB1,on the EMT process of bronchial epithelial cells induced by TGF-?1.6.LncRNA PTTG3P acts as ce RNA,through competitive binding with mi R-192-3p,thus targeting regulation of CCNB1,further promotes bronchial epithelial cell EMT,to participate in the occurrence and development of asthma.