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The mechanism of adhesion molecules-CD44 in asthmatic rat lymphocytesObjective To investigate the mechanism of CD44,a kind of adhesion molecules,in asthmatic rat lymphocytes.Methods The rats asthma models were established by sensitization and challenge with ovalbumin(OVA).After the successful establishment of asthma models,splenic lymphocytes were isolated from rats of the control and asthmatic group.Lymphocytes isolated from asthmatic rats were divided into asthmatic cell(AC)group,CD44 monoclonal antibody(CD44 mAb)group and OxPAPC(TLR2/4 inhibitor)group.Lymphocytes isolated from normal rats of the control group were set up as normal cell(NC)group.CD44 monoclonal antibody and OxPAPC were added into the medium of CD44 mAb group and OxPAPC group respectively.CD44,TLR2,TLR4 and NF-?B mRNA expression were detected by RT-PCR method.The protein expression of CD44 and NF-?B were detected with Western blot method.Results 1.There was significant inflammatory cell infiltration in asthmatic lung tissues compared with lung tissues of normal rats.2.RT-PCR results showed that CD44,TLR4,NF-?Bp65 mRNA expression in asthmatic rat lymphocytes(1.504±0.240,2.102±0.640,1.654±0.260,respectively)were significantly increased compared with those in the control group(0.9974±0.158,1.248± 0.688,1.025 ±0.198),P<0.05,the difference was statistically significant.TLR2 mRNA expression of asthmatic lymphocytes(0.326±0.096)was significantly decreased compared with that of normal control group(0.952±0.258),P<0.01,the difference was statistically significant.TLR4 mRNA expression of CD44 mAb group and OxPAPC group(1.147±0.365,1.223±0.535,respectively)were significantly lower than AC group(2.102±0.640),P<0.01,the difference was statistically significant.The expression of NF-?B mRNA of CD44 mAb group and OxPAPC group(1.064±0.328,0.951±0.250,respectively)compared with AC group(1.654±0.260)were decreased significantly,P<0.01,the difference was statistically significant.3.Western blot results showed that asthmatic CD44 and NF-?B p65 protein expression(1.507±0.215,1.745±0.203,respectively)were significantly increased compared with control group(1.002±0.078,1.012±0.091),P<0.01,the difference was statistically significant.NF-?B p65 protein expression of CD44 mAb group and OxPAPC group(1.101±0.195,1.193±0.117)decreased significantly compared with asthma group(1.745±0.203),P<0.01,the difference was statistically significant.Conclusion CD44 monoclonal antibody can inhibit the expression of TLR4 and NF-?B.CD44 play an important role in promoting asthmatic inflammation through TLR4-NF-?B pathway in rat lymphocytes.Part ? MiR-31 regulates the transcription of adhesion molecules,CD44Objective:To screen and identify the miRNAs that can regulate gene expression of CD44 by targeting CD44 promoter.Methods:1.Culturing human bronchial epithelial cells(BEAS-2b),using bioinformatics analysis to screen out the miRNAs that can target CD44 gene promoter,and overexpressed and konckdowned these miRNAs respectively in human BEAS-2b,48h later,using RT-PCR,Western Blot and cell immunofluorescence method to detect the expression of CD44 in nucleic acid and protein levels,and the supernatants were collected to assess the expression of asthma-related cytokine IL-6,IL-8,ICAM-1 by ELISA.2.Dual luciferase reporter was performed to analyze gene-binding sites when the validated results have suggested that miR-31 target CD44 gene promoter.Samples were divided into 6 groups:promoter-NC(CD44-nc)and miRNA-NC(has-mir-31-nc)were transfected into 293T cells;promoter-NC(CD44-nc)and miRNA(has-mir-31)were transfected into 293T cells;promoter(CD44)and miRNA-NC(has-mir-31-nc)were transfected into 293T cells;promoter(CD44)and miRNA(has-mir-31)were transfected into 293T cells;promoter-M(CD44-mu)and miRNA-NC(has-mir-31-nc)were transfected into 293T cells;promoter-M(CD44-mu)and miRNA(has-mir-31)were transfected into 293T cells.3.BEAS-2b cells were transfected with dsControl or miR-31,chromatin immunoprecipitation(ChIP)assays were performed by using an RNA polymerase ?(RNAp?)-specific antibody to pull down the DNA fragments of CD44 promoter,with primer sets specific to the transcriptional start sites of CD44,the purified DNA was amplified by PCR.Under the intervention of miR-31,we compare the difference of enrichment of RNApII at miR-31-targeted promoters between the experimental group and the control group.Results:1.After 293T cells were transfected within 48h,the luciferase results in each group:experimental group 1,2,3,4,5,6 Firefly/Renilla ratio was 1.000±0.0063?1.170±0.1148?1.000±0.0340?1.235±0.0680?1.000?0.0685?0.971 ±0.0590.Experimental group 1VS experimental group 2,P>0.05,experimental group 3VS experimental group 4,P<0.05,experimental group 5VS experimental group 6,P>0.05,experimental group 4VS experimental group 6,P<0.01,the relative luciferase activity of group 4 were significantly higher than group 3 and 6,and the difference were statistically significant(P<0.05).Compared with the control group,the relative expression of CD44 mRNA in BEAS-2b cells increased after miR-31 mimics was transfected(P<0.05),and when miR-31 inhibitors was transfected,the relative expression was decreased(P<0.05).2.MiR-31 targeted CD44 gene,and the overexpression of miR-31 in BEAS-2b can up-regulate the expression of CD44 and asthma related cytokines,knockdowning miR-31 can contribute to the downregulation of the expression of CD44 and asthma related cytokines.3.ChIP assays have indicated that three primers of CD44 promoter were used to detect the amount of DNA of CD44 promoter region,binding RNApII,in the miR-31 transfected group and the control group,the results suggested that there were significant differences between the experimental group and the control group,and the results of the three primers were consistent.Conclusion:MiR-31 targeted adhesion molecule CD44 and regulated its gene expression at the transcriptional level of CD44 promoter.MiR-31 can regulate the expression of asthma related cytokines ICAM-1,IL-8 and IL-6 by regulating the expression of CD44.Part ? The transcriptional regulation of HD AC inhibitors on CD44 by promotersObjectives To investigate the transcriptional regulation of histone deacetylase(HD AC)inhibitors on CD44 by promoters,and to observe the role of histone acetyltransferases(HATs)and HDACs in asthma.Methods 1.About cells:cultured A549 cells and constructed the target gene CD44 promoter plasmids as the vectors and transfected them into cells.After 24h,add trichostatin A(TSA)respectively with the concentration of 1?M?2.5?M?5?M and sodium butyrate(SB)with the concentration of 1mM?2.5mM?5mM into each group cells.DMSO was served as negative control,and the relative activity of CD44 promoter plasmid luciferase was detected in the case of different concentrations of TSA and SB.Quantitative reverse transcription PCR method(qRT-PCR)was used to detect the expression of target gene CD44 mRNA.2.About mice:32 BALB/c mice were randomly divided into four groups:control group,asthma group,hormone therapy group and TSA treatment group,each group had 8.A mouse model of asthma was established through intraperitoneal injection and atomization inhalation of ovalbumin and control group with equal saline alternative injection and atomization inhalation,hormone treatment group and TSA treatment group mice were given intraperitoneal injection of dexamethasone 1.0 mg/kg(0.2ml)and TSA 1.0 mg/kg(0.2 ml)30min before each challenge.24h after the last challenge,enzyme linked immunosorbent assay(ELISA)was used to measure the levels of cytokines IL-4,IL-8 and IFN-in peripheral blood serum of each group and HAT and HDAC activity in mononuclear cells were measured by enzyme-linked fluorescent assay(ELFA).Results 1.CD44 promoter luciferase activity significantly decreased with the increasing concentrations of TSA and SB in A549 cells compared with the negative control group,suggesting that both TSA and SB could down regulate the CD44 promoter activity.2.The expression levels of CD44 mRNA remarkably decreased with the increasing concentrations of TSA and SB in A549 cells.3.IL-4 and IL-8 levels in asthma group were higher compared with the control group,hormone treatment group and TSA treatment group(P<0.05),and when the control group,hormone treatment group and TSA treatment group compered with each other,the differences were not statistically significant(P>0.05),and there was no significant difference in the level of IFN-in each group(P>0.05).4.Compared with the control group,hormone therapy group and TSA treatment group,HDAC activity in asthma group was lower(P<0.05),however,asthma group HAT activity was significantly higher than the hormone therapy group(P<0.05),and the differences of HDAC and HAT activities in the control group,hormone therapy group and the TSA treatment group were not statistically significant(P>0.05).Conclusions TSA and SB can affect the expression of target gene CD44 at the transcriptional level,and by promoter regulated the transcription of adhesion molecule CD44.The decrease of HDAC activity was associated with the incidence of asthma and also caused the increase of inflammatory factors,which led to asthma.Part IV Expression of microRNA-31 and adhesion molecule-CD44 in serum of children with asthmaObjective To investigate the expression of microRNA(miRNA)-31 in the serum of children with asthma in acute stage and remission stage,and to study the correlation between miRNA-31 and adhesion molecule CD44 in serum of children with asthma.Methods Serum samples were collected from children,including 16 cases of children with asthma in acute attack stage,10 cases in remission stage,and 36 cases of normal children.The total RNA was extracted from these samples and the expression of miR-31 and CD44 were detected by real-time fluorescence quantitative polymerase chain reaction(RT-PCR)method.Results The expression of miR-31 mRNA in the serum of children with asthma during acute attack was significantly higher than that in non-acute attack stage and normal control children,and the expression was positively correlated with the expression of CD44 in the serum of children with asthma.Conclusions The expression of miR-31 was positively correlated with the expression of CD44 in the serum of asthmatic children,and the abnormal expression was involved in the occurrence and development of childhood asthma. |
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