| Background and objectiveSilicosis is an interstitial pulmonary disease induced by long-term exposure to dust particles.Although the pathogenesis of silicosis is unclear,many studies have indicated that genes selective expression controlled by transcription factors(TFs)may contribute to the activation of fibroblasts,which correlated with multi-type cells and cytokines after macrophages swallowed silica.As a result,transwell was used to construct a co-culture model of lung fibroblast and macrophages for observing SiO2-induced differentiation process in vitro.In order to provide reference for the research of lung fibroblast transdifferentiation,RNA-seq and bioinformatics were used to screen the hub TFs and explore the relevant mechanisms.Methods1.Identification the hub transcription factors in the differentiation of lung fibroblasts1.1 The observation of SiO2-induced transdifferentiation model in vitroA vitro co-culture model was constructed,which mice macrophages RAW264.7 phagocytosis SiO2 and induce mice pulmonary fibroblasts NIH/3T3 transdifferentiation.To identify the best silica concentration of phagocytosis,cell viability of RAW264.7 cells exposure to silica was detected by CCK-8 assay.The levels of TGF-β1 were measured by Elisa at the different concentrations of silica swallowed by RAW264.7,According to the results of CCK-8 and Elisa.The experiment was divided into a blank control group,SiO2 exposure group,and TGF-β1.The expression of transdifferentiation markers α-SMA,COL1A1,and FN1 were detected after SiO2 stimulation.1.2 Screening the hub transcription factors in the differentiation of lung fibroblastsDifferentially expressed genes were screened by RNA-seq,and annotated by GO and KEGG database.The expression gene chip datasets that TGF-β1 stimulated IMR-90,MRC-5 and HLF cells were GEO database.The hub TFs obtained from the above results were blasted with AnimalTFDB database.qPCR was used to observe the expression of these TFs after transdifferentiation.1.3 The influence of PPARy/LXRa axis to transdiffitiation of fibroblastsLXRa overexpressed plasmids or siR-LXRa was transfected into SiO2 and TGF-1 induced vitro transdifferentiation co-culture model,respectively.The expression levels of COL1A1 FN1 and LXRa were detected by qPCR and Western Blot.The activity of LXRa in the region of α-SMA promoter was detected by the double luciferase gene report.Cell viability of NIH/3T3 cells exposure to rosiglitazone(RSG)was detected by CCK-8 assay.qPCR was used to detect the expression of α-SMA,COL1A1,and LXRα after giving RSG in SiO2 and TGF-β1 induced vitro transdifferentiation co-culture model.2.The expression of PPARy/LXRa axis in experimental silicosisSixty male C57 mice were randomly divided into control and silica-treated groups.Ten mice per group were sacrificed after they were instilled with silica in the times of 1,7,14,28,and 56 days.HE and Masson staining were used to observe the pathological changes and collagen deposition.qPCR and immunofluorescence were used to detect α-SMA,COL1A1,VIM,PPARy,and LXRa change and identify model.3.Statistical analysisSPSS21.0 software was used to analyze the experimental data.The results under normal distribution were expressed as the mean ±standard deviation,t-test was used when comparing the differences between any two groups.The differences between multiple samples were analyzed by ANOVA.Inspection level of alpha=0.05.Results1.The observation of SiO2-induced transdifferentiation model in vitro and screening the hub transcription factors in the differentiation of lung fibroblasts1.1 The observation of SiO2-induced transdifferentiation model in vitroThe viability of RAW264.7 was constantly decreased after being exposed to SiO2.Compared with the 0～75μg/ml group,there was significant cytotoxicity in the 100 μg/ml group(P<0.05).Compared with the 0～75μg/ml group and the 125μg/ml group,the concentration of TGF-β1 in the cell supernatant was the highest 24h 48 h and 72 h after being exposed to SiO2(P<0.05).Comparing with the control group,α-SMA and FN1 were significantly increased(P<0.05).The expression of COL1A1 was increased after being exposed to SiO2 at 48 h(P<0.05).The expression of α-SMA,COL1A1,and FN1 were significantly increased after stimulated by TGF-β1(P<0.05).1.2 Screening the hub transcription factors in the differentiation of lung fibroblastsThe results of hub TFs were supported by four comparisons and obtained from a BLAST tool from the AnimalTFDB3.0 database at the same time.We found that EGR2 and BHLHE40 were upregulated in fibroblast activation,but TBX2,NR1H3,NR2F1,PPARG,and EPAS1 were downregulated in the process that fibroblast towards myofibroblast.The expression of seven hub TFs were consistent in the cells of HLF,MRC-5,IMR-90,and NIH/3T3,suggesting that these seven hub TFs and PPARG/LXRa axis might be of great importance in the trans-differentiation of lung fibroblast.1.3 The influence of PPARγ/LXRα axis to transdiffitiation of fibroblastsCompared with siR-NC group,LXRα was significantly decreased after being transfected of siR-LXRα(P<0.05),but α-SMA and COL1A1 were upregulated after stimulated by SiO2 and TGF-β1(P<0.05).Compared with siR-NC group empty plasmid group,LXRα was significantly increased after being transfected of pEGFP-LXRα(P<0.05),but α-SMA and COL1A1 were upregulated after stimulated by SiO2 and TGF-β1(P<0.05).The result of Dual-Luciferase Reporter Assay revealed that the luciferase activity level of Acta2-WT+LXRα-NC group was higher than Acta2-NC+LXRα-NC group(P<0.05),Acta2-WT+LXRα group was lower than Acta2-WT+LXRα-NC group(P<0.05),and Acta2-WT+LXRα group was lower than Acta2-Mut+LXRα group(P<0.05).Compared to the DMSO control,the viability of NIH/3T3 was initially inhibited by 30 μmol/L RSG,which was used the subsequent dose concentration.Compared with NC group,LXRα was decreased after adding RSG(P<0.05),but α-SMA was no significant difference.In addition,compared to DMSO group,LXRa was significantly increased but α-SMA(P<0.05)was downregulated after adding RSG(P<0.05).2.The expression of PPARγ/LXRa axis in experimental silicosisThe alveolar wall became widened and plenty of inflammatory cells infiltrated around the small airway after being exposed to SiO2 for 7 days.The degree of inflammatory infiltration reached the highest,collagen fiber deposition in alveolar cavity increased significantly,and there were obvious cellular nodules after being exposed to SiO2 for 28 days.The number of cellular nodules increased and multiple areas of dense fibrous deposition were observed after being exposed to SiO2 for 56 days,but no obvious fibrous nodules were observed in lung tissue.The expression ofα-SMA was no significant change in the times of 1,7,and 28 days,but increased in 56 days(P<0.05).The expression of PPARy was downregulated in the times of 14,28,and 56 days(P<0.05),but no significant change in the times of 1 and 7 days.The expression of LXRa was downregulated in the times of 1,14,and 56 days(P<0.05),but no significant change in the times of 7 and 28 days.ConclusionsMultiple TFs related to SiO2-induced transdifferentiation of lung fibroblasts,among which PPARγ/LXRα axis was downregulated in silicosis and might be the potentially effective target for silicosis. |
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