Preliminary Study On The Role Of MiR-149in Silica-induced Pulmonary Fibrosis

Coal workers’ pneumoconiosis（CWP） is characterized by irreversible lungfibrotic diseases resulting from chronic dust inhalation（silica,silica and coal or coaldust）, which mainly includes the silicosis and coal pneumoconiosis. CWP is one ofthe most serious occupational disease in China with no effective therapies. Currentlythe pathogenesis of silicosis is still unknow. The numerous studies have shown thatthe processes of pulmonary fibrosis include inflammatory injury, tissue desdructionand abnormal tissue repair with matrix deposition. The pathological features showincreasing fibroblast proliferation and collagen synthesis in the lung tissue. And itinvolves in a large number of inflammatory cytokines and fibrosis factor expressionand regulation of related genes in complex networks. Recently, microRNAs（miRNAs） as small noncoding RNA molecules of18²2nucleotides （nt） in length,play critical roles in various physiological processes such as inflammation, tissuedevelopment and differentiation, cellular apoptosis, proliferation, and tissue repair,which have emerged in the recent decade as key regulators of organ phenotypes andpotential targets for therapeutic interventions in multiple diseases. But there are fewstudies refer to miRNAs in the research of silicosis. Considering the prevalence forsilicosis in China, the present study was about the miRNA screening in serum ofCWP patients and healthy controls, and further functional studies both in vivo and in vitro were also conducted on the candidate miRNA. Hoping to find out thebiomarkers of diagnosis, progression and treatment of CWP, and further provide cluesto reveal the pathogenesis of CWP/silicosis and the roles for certain miRNAs in it.The dissertation is divided into two parts: analysis of serum genome-widemiRNAs for CWP in a case-control study and preliminary study of the role formiR-149in silica-induced pulmonary fibrosis.Part â… : Analysis of Serum Genome-wide miRNAs for CWP in a Case-ControlStudy.Objective: The key miRNAs which involved in pathogenesis and development ofCWP will be explored by screening the different expression of miRNAs in serumbetween cases and controls, so as to provide further clues to pathogenesis and newtherapies of pneumoconiosis.Methods:（1） The epidemiological information of the research subjects were obtainedby questionnaire, which included age, exposure history, jobs and other relatedepidemiological items. Health examinations including high KV chest X-ray and DRwere conducted by occupational professional agency. CWP patients were diagnosedaccording to the China national criteria for pneumoconiosis （GBZ70-2009）.One control group and three case groups which were CWP stage â… , stage â…¡ and stageâ…¢ （n=9in each group） were mainly matched by age, exposure year, job, diseasehistory, smoking and so on.（2） Equal volume of sera from cases and controls in eachgroup were pooled for SOLiD sequencing-based miRNA profiling.（3） The keymiRNA target genes were primarily selected according to miRNA profiling resultsthat were blast to the known miRNA databases to identify the differently expressedmiRNAs with2-folds change, in which candidate miRNAs with the accordant changetrends in all of the case groups compared to the controls were identified. Genespotentially targeted by the selected miRNAs were identified by using the miRWalkdatabases available online.Results: Compared to the miRBase （15.0）,39.9%、42.2%、36.4%、39.9%knownprecursor miRNAs were identified in control, stage I, stage II and stage III of CWP, respectively. In these effective reads, the most abundant length was22nt size class.Using2-fold change as a cutoff level,32,55and39deregulated miRNAs wereidentified in each stage cases compared to controls, in which two up regulatedmiRNAs and three down regulated miRNAs with an accordant change in all of thecase groups compared to the paired controls were identified （miR-25*,miR-9*upregulated; miR-20b,miR-222and miR-149down regulated）. Target genes predictionand pathway analysis were then conducted for the candidate miRNAs, suggesting thatthe differentially expressed miRNAs regulate proliferation, apoptosis, inflammation,cell cycles, gene transcription and other biological process. Pathway analysis hasshown that candidate miRNAs regulated the key pathways in process of pulmonaryfibosis.Conclusion: Totally two up regulated and three down regulated miRNAs with anaccordant change direction were identified in all of case groups compared to thepaired control. And the candidate miRNA molecules may be involved in the keyprocesses for pulmonary fibrosis.Part â…¡: Preliminary Study on the Role of miR-149in Silica-induced PulmonaryFibrosis.Objective: Basing on the miRNA profile results, to explore the potential roles ofmiR-149in the pathogenesis of silica-induced pulmonary fibrosis.Methods: Studies were conducted both in vivo and in vitro to verify the preliminaryresults and to find new mechanisms of miR-149in regulating silica-inducedpulmonary fibrosis.（1） In vivo studies: The expression of miR-149was tested byReal-time PCR and inflammatory cytokine IL-6by immunohistochemistry andWestern blot assay in the lung tissues of silica-induced pulmonary fibrosis mice,.（2）In vitro studies:â‘ The m odels ofSiO₂exposed cells （A549and HBE） wereestablished. The A549cells were transfected with miR-149mimics and inhibitor, andthen the expression of IL-6in cells was tested by Western blot assay;â‘¡T heexpression of IL-6in CWP patients and paired controls was determined byEnzyme-linked immunosorbent assay. Results:（1） The serum expressions of miR-149in each stage of CWP patients weredown regulated compared to that of controls, but no significant differance was foundin expression of IL-6. However, it was found that the expression of IL-6was upregulated with the progress of CWP.（2） In the mice model, the pulmonary alveoli andinterstitial were infiltrated with amount of inflammatory cells after treated in week1in both silica-treated and saline-treated groups. The inflammation alleviatedgradually in saline-treated mice in2weeks, and was similar to that of controls. Butfor the silica-treated mice, the pulmonary alveolar wall were destoried and interstitialthickened obviously gradually. The expression of miR-149in lung tissues downregulated significantly in any check points（P<0.000）, while IL-6expression was upregulated obviously.（3） In the silica-treated cell models, the apoptosis of A549andHBE cells increased after silica-treated, especially for A549cells which was moresensitive to the silica challenge. For the A549cells, the expression of IL-6increasedin a silica-treated dose and time-dependant manner, while HBE cells only showsilica-treated dose-dependant effect. In addition, we found a significantly decrease formiR-149in HBE cells after12hours treated with the150μg/ml of silica（P<0.000）,the expression of miR-149was almost totally inhibited by the silica exposure, whileno significance was found inA549cells after24hours treated with the50μg/ml ofsilica（P=0.234）. But a decreased trend can be found in it. At last, we verified that thedysregulated expression of miR-149with miR-149mimics and inhibitor transfectioninfluenced the IL-6expression in A549cells. High expression of miR-149inhibitedIL-6expression. Reverse results were found after miR-149was inhibited.Conclusion:（1） Inflammatory reaction was induced after silica exposure in mice;（2）Down regulation of miR-149and up regulation of IL-6may involved in theinflammation and fibrosis process of silica-induced pulmonary fibrosis;（3） miR-149can inhibit IL-6expression in A549cells.