A Preliminary Study On MicroRNA Screening For Differential Expression Of Pneumoconiosis

?Objective?The miRNAs closely related to the development of silicosis were screened and verified by analyzing changes in miRNA expression profiles in lung tissue at different stages of silicosis animal models.The differentially expressed miRNAs were further validated at the cellular level and in the serum of silicosis patients,and the application value of miRNAs as early biomarkers of silicosis was explored.?Methods?Male Wistar rats were randomly divided into control group and silica?SiO₂?dusting group,6 rats in each group.The lung tissues of each group were collected at corresponding time points after dusting,and 3 rats were taken out of lung tissue.For pathological observation,the other three miRNA microarray technology were used to screen differentially expressed miRNAs in lung tissues.The expression levels of differentially expressed miRNAs in rat lung tissues were verified by real-time PCR?RT-qPCR?.Target gene prediction was performed on differentially expressed miRNAs in both groups,and GO enrichment analysis and KEGG pathway analysis were performed.On the basis of previous experiments,A549 cells were cultured in vitro at the cell level,and divided into control group,SiO₂ stimulation group and TGF-?1stimulation group.RT-qPCR method was used to verify the expression level of miRNA in A549 cells,and then choose occupational health in Shandong Province.18patients with stage I,II and III silicosis in the Occupational Disease Prevention and Treatment Research Institute,and 18 healthy checklists in a healthy control group were male.The RT-qPCR method was used to verify miRNA in silicosis patients and healthy controls.The level of expression in serum.The results of measurement data and semi-quantitative data were analyzed by SPSS 22.0 statistical software.?Results?1.General conditions of animalsRats in the model group developed shortness of breath and slow weight gain.On the 28th day after model establishment,the lung tissues of the control group were normal pink,normal size and good elasticity.The rats in the model group were white,obviously enlarged,and had poor elasticity.Gray-white nodular lesions were visible to the naked eye.There is a feeling of sand in the touch.After the model establishment,the body weight of the two groups increased.The weight growth rate of the model group was significantly lower than that of the control group on the 14th,21st and 28th day.The 21st and 28th model groups were statistically different from the control group?P<0.05?.2.Histopathological examinationLight microscopy showed that the HE tissue of the control group showed clear lung structure,HE staining at 1,7,and 14 days showed that neutrophils were mainly infiltrated by neutrophils in the lung tissue of the dust-treated group,28d HE staining showed that the alveolar structure of the lung tissue was unclear,and a small amount of necrotic cells were observed locally,and the fibroblasts were significantly increased.No obvious collagen fibers were found in the control group and the 1,7,14d dusting group.The masson staining showed that the lung interstitial of the rats in the dust-bleached group showed large-thick collagen fibers deposited in the local granulation.Collagen proliferation around the tissues,bronchi and blood vessel walls is evident.3.Detection of miRNA microarray in lung tissueCompared with the miRNA microarray results of the lung tissue of the control group,the miRNAs were screened for 144 different miRNAs,63 of which were significantly up-regulated and 81 of which were significantly down-regulated.Differentially expressed miRNAs may regulate gene expression associated with inflammation,gene transcription,cell cycle,and signal transduction.The function of differential miRNA target genes is mainly related to protein binding,apoptosis process and transcription of DNA as a template,and mainly plays a role in cytoplasm and extracellular bodies.Moreover,related target genes play a role in the development of pulmonary fibrosis by participating in the TGF-?and PI3K-AKT signaling pathways,which affect cell proliferation and apoptosis.4.RT-qPCR verification miRNA chip resultsThe miR-29b-3p,miR-34c-3p,let-7d-3p,miR-672-5p,miR-582-3p and miR-26a-5p were down-regulated in the microarray,and the expression was up-regulated.The miR-21-3p,miR-423-5p,miR-3544,miR-542-3p,miR-155-5p and miR-146-5p were verified by RT-qPCR,compared with the control group,in the dusting group.The expression levels of miR-29b-3p,miR-34c-3p,let-7d-3p and miR-26a-5p were significantly down-regulated?P<0.05?,and the expression levels of miR-21-5p and miR-423-5p were significantly up-regulated?P<0.05?.5.Relative expression of miRNAs in A549 cellsThe cells were harvested after 12,24 and 48h,and the total RNA was extracted for RT-qPCR.Compared with the 12h group,the expression levels of miR-29b-3p and miR-34c-3p were significantly down-regulated in the 24 and 48h groups,The expression level of miR-155-5p was significantly up-regulated,and the difference was statistically significant?P<0.05?.6.Lung ventilation function test and serum miRNA expression analysisThe four indicators were all abnormal in stage II and stage III silicosis,and were normal in the control group and stage I silicosis.RT-qPCR showed that the expression of miR-155-5p and miR-21-5p in the case group was higher than that in the control group?P<0.05?,and the expression of miR-26a-5p,miR-34c-3p and miR-29b-3p in the case group was lower than that in the control group?P<0.05?.And the expression level of miR-21-5p was increased in the control group and in stage I,II,and III silicosis,and the expression levels of miR-34c-3p and miR-29b-3p were decreased in the control group and in stage I,II,and III silicosis.?Conclusions?1.Six miRNAs closely related to the development of pulmonary fibrosis were found in the animal model of the dust-exposed rat.The up-regulated miRNAs were miR-21-5p and miR-423-5p,and the down-regulated miRNAs were miR-29b-3p.miR-34c-3p,let-7d-3p and miR-26a-5p,differentially expressed miRNAs regulate genes involved in inflammation,apoptosis,cell cycle,gene transcription and signal transduction.2.The expression levels of miR-29b-3p and miR-34c-3p were significantly down-regulated in SiO₂ and TGF-?1-treated A549 cells,miR-155-5p expression level was significantly up-regulated.This suggests that these three genes may regulate the development of pulmonary fibrosis.The expression level of miR-21-5p in the serum of silicosis patients increased in the control group and in the silicosis of stage I,II and III.The expression levels of miR-34c-3p and miR-29b-3p were decreased in the control group and in stage I,II,and III silicosis,these three miRNAs may indicate the progression of the disease.3.The expression level of miR-21-5p was significantly up-regulated in the serum of silicotic rats and silicosis patients.The expression levels of miR-29b-3p and miR-34c-3p were significantly down-regulated in the serum of silicotic rats,sputum A549 cells and silicosis patients.These three miRNAs may regulate the development of pulmonary fibrosis and may be an indicator of early silicosis diagnosis and prognosis.