The Mechanisms Of MicroRNA Regulating The Expression Of Alveolar Macrophage TLR2 Receptor In COPD Rat

BackgroundRecently, with a rising incidence, invasive pulmonary aspergillus (IPA) is closely related to the human life-time extension, tumor chemotherapy, organ transplantation, using of broad-spectrum antibiotic, cortical hormone and immunosuppressor. It was suggested that cause of this disease may be immunosuppressor. However, more and more evidence showed that patients without immunosuppressor but COPD, may have high risk of IPA. Our previous research found that the incidence of IPA in COPD patients was 3.9% while the death rate reached 48%.Currently the increased IPA susceptibility in COPD patients become a hot area of research, one of the main reasons of which may be dysfunction of respiratory system. The internal immunological function in alveolar macrophage plays the important role during IPA defense which was demonstrated under smoke exposure, meanwhile little of molecular mechanism was known till now. alveolar macrophage can recognize and adhere aspergillus spores, which activate immune and inflammation response. TLR2, one of the most important PRRs, can recognize composition of aspergillus cytoderm and then release the inflammatory factors through activated NF-κB.Our previous results suggested that, COPD rats had a low expression of TLR2 in alveolar macrophage whose up-regulation was inhibited after aspergillus spores transplantation, with respect of healthy rats. We also found the mRNA level of TLR2 was increased in alveolar macrophage from COPD rats with aspergillus spores transplantation. Thus it is implied that smoke may result in unusual up-regulation of TLR2 in alveolar macrophage when inflammation stimulation was activated, which may damage the immune function without any effect on TLR2 gene transcription.MiRNAs are a large group of short non-coding RNAs known to be involved in the post-transcriptional regulation through degradation of targeted mRNA or by blocking protein translation through binding into 3’UTR region, which may facilitate the further understanding of the disease process. As one of early responses when facing exogenous or endogenous signal, miRNA expression plays a significant role in immune cells during the whole development process and immune response. Till now a lot of research demonstrated that smoking may affect the expression of some miRNAs in lung tissue. Thus we conferred that there are some miRNAs with alternation in COPD patients who use cigarette for a long time, which may result in abnormal inflammatory response in alveolar macrophage. These miRNAs may regulate the inflammatory response with positive or negative feed back. Some miRNAs may be involved in activating some signaling pathways and regulating TLR2 expression, which may injury the immune function of alveolar macrophage.In our study, we investigated the differentiate miRNA in alveolar macrophage from COPD rats and healthy rats respectively as detected by miRNA microarray, and then screen out the potential target gene which may be involved in TLR2 regulation using bioinformatics assay and dual-luciferase assay. Finally we explored the molecular mechanism of effect of miRNA on TLR2 in COPD rats which may help to further investigate the effect of smoking on immune function in alveolar macrophage.Chapter I screening of differentiate miRNAs in alveolar macrophage from COPD ratsPurpose:to screen out the differentiate miRNAs in alveolar macrophage from COPD rats using miRNA microarray assay, which may provide evidence for further investigation.Methods:30 female Wistar rats were divided into 2 groups as follows:(A) COPD model group:smoking was conducted in a home-made smoking box and endotoxin tracheal instillation were conducted twice a day for 90 days; (B) Control group:no smoking but saline tracheal instillation were conducted for 90 days. COPD model was verified by lung tissue CT, lung function test and lung tissue pathology, alveolar macrophages were verified by Wright’s staining and immunofluorescence staining. Blood and tissues of 3 rats from both of groups were collected and stored at -80℃ (less than a week) before they were tested. Total RNAs were extracted from alveolar macrophages whose quality and integrity were evaluated using spectrophotometer and gel electrophoresis. Then miRNAs expression was marked and then detected in both groups by microarray assay and chips assay using kits purchased from Exiqon (Danmark).Results:result of lung tissue CT, lung function test and lung tissue pathology was in accordance with COPD onset. Wright’s staining result showed that cells were round or ellipse with big and stained nuclear. CD68 was positive in alveolar macrophages by immunofluorescence staining. Compared to control group, let-7b-3p, miR-675-5p and miR-376c-3p were decreased in COPD model group without any up-regulated miRNAs. Iet-7b-3p and miR-675-5p were inhibited significantly in COPD model group Ⅱ while miR-200b-3p, miR-665, miR-344b-1-3p, miR-34c-5p, miR-34b-5p, miR-99b-5p, miR-129-1-3p, miR-3557-5p, miR-331-5p, miR-493-5p and miR-200a-3p11 were increased accordingly.Conclusion:1) COPD rat model was generated successfully; 2)compared to healthy rats, many miRNAs expression differently in alveolar macrophages from COPD rats, which was agreeable with trends in human alveolar macrophage. It is suggested that alternative miRNAs in alveolar macrophages may be related to inflammatory response.Chapter Ⅱ prediction of miRNAs regulating TLR2 expression in alveolar macrophage from COPD ratsPurpose:to screen out the miRNA which could regulate the TLR2 expression in alveolar macrophages from COPD ratsMethods:target genes of 13 screening miRNAs described in Chapter I were predicted using miRbase, miRanda and miRdb, result of which was used for cross check with TLR2. To avoid any missing information, we amplified the searching field. We also predicted reversely the target miRNA of TLR2 and tried to find out the potential miRNA beased on publications using NCBI website. Then we cross checked and compared all miRNA candidates (including those without significant difference) based on both prediction analysis and microarray analysis. Finally we got all potential miRNAs which may regulate TLR2 in alveolar macrophages from COPD rats through the three ways above. After exclusion of false positive result of microarray assay, real-time PCR was used for validation. Finally, DAVID 6.7 system was used for Gene Ontology and KEGG pathway analysis based on the positive screening result.Results:12368 target genes were predicted through miRbase, miRanda and miRDB. Only result from miRDB suggested the target gene of miR344b-1-3p was TLR2. then we chose miRWalk database and reversely predicted 11 miRNAs which may bind into TLR2. after cross checking with microarray result, it is suggested that Rno-miR-124-3P, rno-miR-363-5P and rno-miR-9a-3p expressed positively in microarray assay. Based on NCBI reference searching, we found that TLR2 may be the target gene of miR125, miRNA 19a/b and miRNA146a. combined with microarray result, it was implied that rno-miR-125b-5p, rno-miR-19b-3p and miRNA 146a-5p had expression. All 7 miRNAs mentioned above were validated by qPCR with amplified samples, among which, rno-miR344b-1-3p, rno-miR-125b-5p and miRNA 146a-5p expressed with significant difference. Except rno-miR-125b-5p, result of all other 6 miRNAs in microarray was in accordance with which in qPCR. Expression of miR-344b-1-3p was increased with significant difference in both microarray and qPCR analysis. KEGG signaling pathway enrichment analysis showed that, target gene of miR-344b-1-3p was related to TLR signaling pathway.Conclusion:1) result of microarray was validated by real-time PCR which suggested that microarray assay is reliable; 2) miR-344b-1-3p was picked out which could regulate the TLR2 expression in alveolar macrophages from COPD rats; 3) pathway assay for miR-344b-1-3p target gene showed that effect of miR-344b-1-3p may be related to TLR signaling pathway.Chapter Ⅲ effect of miR-344b-1-3p on TLR2 in alveolar macrophage as detected by dual-luciferase assayPurpose:to verify if miR-344b-1-3p can target the 3’UTR region of TLR2 directly Methods:bioinformatics analysis was applied for prediction of miR-344b-1-3p binding location in TLR2 gene through online analysis software. TLR2-3’UTR region was synthesized via sequence splicing using long primer and point mutation.The target gene TLR2-3’UTR region was sub cloned forwardly and reversely and the multiple cloning site (MCS), located downstream of terminal codon, was translated by Renilla Luciferase following by, which was finally inserted into vector and received the sequencing evaluation. This recombinant plasmid was transfected into 293 cell line before conducting dual-luciferase assay.Results:the recombinant plasmid was constructed successfully which contained TLR2 3’UTR region. miR-344b-1-3p could bind into 3’UTR region of TLR2 as detected by dual-luciferase assay, compared to control group, whose result showed that the luciferase activity was decreased (p<0.05). Conclusion:miR-344b-1-3p regulate TLR2 specificallyChapter IV effect of miR-344b-1-3p on TLR2 expression and signaling pathway in alveolar macrophage from COPD ratsPurpose:To explore the impact of microRNA344 b-1-3P on TLR2 expression, involved signaling pathway and secretion of inflammatory factors in alveolar macrophages from COPD rats.Methods:1. The COPD cell model was constructed with exposure of cigarette smoke extraction (CSE), which was divided into four groups:A:NR8383 cells+CSE (5%); B:NR8383 cells+CSE (10%); C:NR8383 cells+CSE (15%); D:Blank control group:NR8383 cells without any treatment. Pre-treated with CSE for 24 hours, apoptosis of NR8383 cells was conducted by flow cytometry and meanwhile miR-344b-1-3p expression was detected by qPCR.2. The recombinant lentivirus was constructed with miR-344b-1-3p inhibitor insert, which was transfected into NR8383 cells.3. To investigate the effect of miR344b-1-3p on TLR2 and related signaling pathway in alveolar macrophages from COPD rats, cells were divided to three groups: A:NR8383 cells+CSE (10%)+recombinant lentivirus+Pam3CSK4; B:NR8383 cells+CSE (10%)+vector+Pam3CSK4; C:NR8383 cells+CSE (10%)+ Pam3CSK4. NR8383 cells were stimulated by CSE (10%) for 24 hours before being transfected with recombinant lentivirus or vector for 24 hours. Next NR8383 cells were treated by lug/ul Pan3CSK4 for 1 hour before harvesting. TLR2 expression was tested through flow cytometry and the mRNA level was evaluated via qPCR after total RNA being isolated. Meanwhile, protein levels of IRAK-1, IκBα, ERK, JNK and p38 were detected by western blot after total protein being collected. At last, the upper clear liquid was collected to identify TNF-a, IL-1β and MIP-2 levels.Results:1. The low apoptosis rate of blank control group (NR8383 cells) was increased dose-dependently with CSE treatment. The NR8383 cell apoptosis induced by CSE treatment was mainly in early phase which was (15.20%,29.93%,49.98% and 52.98%) in blank control group, CSE (5%) group, CSE (10%) group and CSE (15%) group respectively, with significant difference (P<0.05) in pair comparison; the total apoptosis rate of four groups were (22.12%,39.98%,61.98%and 64.32%) respectively with significant difference (P<0.05) in pair comparison. The result of qPCR showed that miR-344b-1-3p expression was increased along with CSE concentration, however, when the CSE concentration achieved 15%, miRNA expression was different but without any statistical difference from which treated with 10% CSE.2. Based on sequencing verification, lentivirus containing miR-344b-1-3p inhibitor was constructed successfully.3. Using flow cytometry, TLR2 expression in group A was higher than group B and group C after Pam3CSK4 and lentivirus treatment.4. Western blotting result showed that expression of TLR2 ligand proteins including IKBa, IRAK-1, ERK, JNK and p38, was increased in group A compared to group B and C. result of gray semi-quantitative analysis showed that the relative expression of TLR2 ligands, normalized with GAPDH, were higher in group A than group B and group C.5. The upper clear liquid concentration was increased in all three groups while TNF-a, IL-1β and MIP-2 levels in group A were higher than group B and C with statistical difference (P<0.05). However, IL-10 expression was lower compared to blank control group without statistical difference (P>0.05).Conclusion:1. miR-344b-1-3p expression was induced in NR8383 cells with treatment of CSE; 2. miR344b-1-3p regulated TLR2 expression through signaling pathway, which may result in the secretion of inflammatory factors.