| Objective Based on a rat model of inhalation experimental silicosis,screene the differential LncRNAs and mRNAs related to experimental silicosis throughRNA-squencing.Investigate the functions and regulatory mechanism the representative differentiallncRNAsincludingLncRNALOC102549714（Lnc714）,LOC103691771（Lnc771）and differential mRNAs,secretory phosphoprotein 1（SPP1）in the process of silicosis fibrosis.Methods The rat model of silicosis was established by moving dust staining system and randomly divided into control 24 weeks group and 24 weeks dust exposure group.The pathological changes of lung tissue were observed by HE stain and sirius red stain,immunohistochemical stain（IHC）and western blot were used to detect the localization and expression of smooth muscle actin（α-SMA）and collagen I（COL I）.The differential LncRNAs and mRNAs related to experimental silicosis were screened byRNA-sequencing.Bioinformatics analysis of candidate differential LncRNAs and mRNAs were performed using GO and KEGG enrichment analysis.Expression of candidate differential LncRNAs[Lnc714,Lnc771,LOC102550137（Lnc137）,LOC103692016（Lnc2016）,LOC103693125（Lnc125）]and mRNAs（SPP1、Mmp12、Ccl7、Defb5、Slc26a4、Fabp4、Lpo、Lcn2、Itln1 and Dlk1）were detected by q RT-PCR in lung tissues of silicosis rats,as well as in TGF-β1-induced lung fibroblasts,Si O2-induced RAW264.7 cell,Si O2-induced MLE-12and the macrophages of bronchoalveolar lavage fluid（BALF）from silicosis patients.Lnc714,Lnc771 and Lnc714+miR-873-3p minic（Lnc714+mi873-3p）were silened or overexpressed in lung fibroblasts,and the expressions of COL I,α-SMA,transforming growth factor type I receptor（TGF-βRI）,TGF-βRII,p-Smad2/3 and Smad2/3 were detected at basal level and TGF-β1 induced level.The content of SPP1 in serum was detected by ELISA.Combined with Establishe Experimental silicosis model,RAW264.7cell model induced by Si O2 in vitro,and SPP1 recombinant protein and Ac-SDKP were used to intervene.SPP1 expression and localization were detected by IHC.The expression of COL I,monocyte chemoattractant protein-1（MCP-1）,TGF-β1,TGF-βRI,TGF-βRII,p-Smad2/3,Smad2/3 and SPP1 in lung tissue and RAW264.7 cells were measured by western blot.Results 1 HE staining,sirius red stain,IHC stain and western blot showed that compared with the control 24 week group,the typical fused cellular fibrous nodules were observed in the 24 week dust exposure group,and there were more red cord collagen fiber deposition,α-SMA and COLI were shown strong positive expression in silicon nodule and surrounding interstitial fibrosis region.High-throughput transcriptome sequencing results indicated that 86 lncRNAs were up-regulated and 59 lncRNAs were down-regulated in silicosis group（P<0.05）.According to the variation ratio and abundance,5 LncRNAs were obtained and q RT-PCR results showed that the expression of LOC103691771,LOC102549714,and LOC102550137 was increased,whereas the expression of LOC103693125 and LOC103692016 was decreased significantly in silicosis samples compared to control samples（P<0.05）.q RT-PCR results showed that Lnc771 and Lnc714were increased in BALF,TGF-β1-induced fibroblast,Si O2-induced RAW264.7macrophages and MLE-12 cells（P<0.05）.IHC and western blot results showed that compared with the control group,the expressions ofα-SMA,COL I,TGF-βRI,TGF-βRII and p-Smad2/3 were inhibited in silence Lnc771 or Lnc714 group.Howerer,the expressions ofα-SMA,COL I,TGF-βRI,TGF-βRII and p-Smad2/3 were increased in overexpression of Lnc771 or Lnc714 group compared with control group（P<0.05）.Mi R-873-3p minic on the basis of overexpression of Lnc714 inhibited the expression ofα-SMA,COL I,TGF-βRI,TGF-βRII and p-Smad2/3 and cell migration in lung fibroblasts.2High-throughput transcriptome sequencing results indicated that 912 mRNAs were up-regulated and 426 mRNAs were down-regulated in silicosis group（P<0.05）.According to the variation ratio and abundance,10 mRNAs were obtained and q RT-PCR results showed that the expression of Spp1,Mmp12,Ccl7,Defb5,Fabp4 and Slc26a4 were increased in silicotic rats（P<0.05）,as well as Lpo,Itln1,Lcn2,Dlk1 were decreased（P<0.05）.Elisa result showed that SPP1 was increased in serum from silicosis patients（P<0.05）.IHC and western blot results showed that compared with the control group,the expressions of COL I,MCP-1,TGF-β1 and SPP1 in the silicosis group were up-regulated,and the expressions of COL I,MCP-1,TGF-β1 and SPP1 in the Ac-SDKP group were decreased compared with the silicosis group（P<0.05）.Compared with the control group,the protein expression levels of COL I,MCP-1,TGF-β1 and SPP1 were up-regulated in the Si O2-induced macrophage.In addition,the expression levels of COL I,MCP-1,TGF-β1 and SPP1 in the Ac-SDKP treated group were down-regulated compared with Si O2-induced macrophage.（P<0.05）.Compared with the control group,the expression of TGF-βRI,TGF-βRII and p-Smad2/3 was up-regulated in the SPP1 induced macropahes.Compared with the SPP1induced macropahes,the expression of TGF-βRI,TGF-βRII and p-Smad2/3 was down-regulated in the Ac-SDKP treated-macrophaes and LY364947 treated-macrophaes（P<0.05）.Conclusions The differential expression of 145 LncRNAs and 1338 mRNAs were found in lung tissue of inhalation silicosis rat,among which Lnc771 and Lnc714 were involved in the regulation of myofibroblast differentiation through TGF-β1/Smad2/3 signaling pathway.The representative differential mRNA SPP1 could activate the TGF-β1 signaling pathway,thus promoting the formation and progression of silicosis fibrosis in rats.Targent blocking of SPP1/TGF-β1 signaling could inhibit the formation and progression of silicosis fibrosis in rats.Figure 43;Table 9;Reference 245... |
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