Study On The Role Of LncRNA MRAK050699 In Silicosis By Regulating EMT Process

Objective By constructing a rat model of silicosis,the expression profile of lncRNA specific to silicosis was observed to simulate the pathogenesis of silicosis.Lnc RNAMRAK050699,in rat alveolar type II epithelial cells（RLE-6TN cells）was knocked out to explore the effect of MRAK050699 on epithelial-mesenchymal transformation（EMT）of RLE-6TN cells induced by SiO₂ dust,and then to observe the regulatory effect of MRAK050699 on the process of silicosis fibrosis.Methods 1.20 male SD rats of SPF grade were randomly divided into two groups with 10 rats in each group:normal saline control group and silicosis model group.50 mg of free SiO₂ dust was injected into the trachea of the model group,and the same amount of sterilized saline was injected into the normal saline control group.Then the treated rats were fed normally for 45 days.Hematoxylin-eosin（HE）staining,Masson（Masson）staining and immunohistochemical（IHC）detection were performed,and real-time fluorescence quantitative polymerase chain reaction（qRT-PCR）was used to detect the screened lncRNA in tissues.The gene chip was prepared to construct the regulatory network of differentially expressed micro RNA-target genes.The up-and down-regulated differentially expressed genes were predicted by mi Randa tool,and sorted according to the differential multiple,and the integrated differentially expressed lncRNA-micro RNA-mRNA network was constructed by cytoscape software.The enrichment pathway of lncRNA-related target genes was analyzed by KEGG,and the function of Lnc RNA-related target genes was analyzed by gene ontology（GO）.Fluorescence in situ hybridization（Fluorescenceinsituhybridization,FISH）was used to observe the localization of lncRNA in cells.2.The effect of SiO₂ dust on the viability of NR8383 cells was determined by CCK8 method.The expression of TGF-βsecreted by NR8383 cells stimulated by different concentrations of dust was detected by Western blotting（Western blot）.The co-culture system was established,and the experiment was divided into four groups:normal group,model group,knockout control group and knockout group.（the ratio of NR8383 cells to RLE-6TN cells was 1:2）.40μg/cm² SiO₂ dust suspension 150μl was added to the transwell chamber of the latter three groups of co-culture system,and the four groups continued to be cultured for 24 hours at the same time.At the end of culture,the transwell chamber was taken out,and the state of the cells in the lower chamber was observed.The total RNA and total protein of RLE-6TN cells in the lower chamber were extracted,and the m RNA and protein expression levels of TGFbr1,p-Smad3,E-cadherin,N-cadherin,α-SMA,Vimentin,Snail,ZEB1,ZEB2,COLⅠ,Col Ⅲ and FN were detected respectively.Results 1.The results of lung tissue anatomy of rats showed that the lungs of rats in the normal saline control group were pink,smooth,soft and elastic,while scattered gray-white plaques and hard plaques could be seen in the lung tissue of rats in the silicosis model group.The results of HE and Masson showed that compared with the model group,the normal saline control group showed clear alveolar structure,slightly dilated blood vessels and hyperemia,and there was no proliferation of collagen fibers in the alveolar septum.In the silicosis model group,the alveolar structure was incomplete,obvious nodular structure,emphysema,patchy fibrosis could be seen,and the distribution of blue collagen filaments and obvious proliferation of collagen in the nodules could be seen in the silicosis model group.The results of immunohistochemistry showed thatα-SMA and COLⅠproteins were highly expressed in the model group.The results of gene chip showed that there were 1077 differentially expressed lncRNAs,of which 378 were up-regulated and 699 were down-regulated.Among the differentially expressed m RNA,215 cases were up-regulated and 510 cases were down-regulated.Tgfbr1 is the most important target gene involved in TGF-βsignaling pathway.The expression level of Lnc RNA in rat lung tissue was verified by PCR technique.The level of Tgfbr1m RNA and the expression of 21 lncRNA were up-regulated in the lung tissue of silicosis model group,and the expression of MRAK052509,MRAK139674,AY539881,MRAK050699,XR＿6113,uc.452+ and BC167061 was the most significant.Spearman test was used to analyze the correlation between the expression of MRAK016357,MRAK052509,S56463,MRAK050699,BC167061,U06752,AY007691 and Tgfbr1 in the lung tissue of silicosis rat model.Using the method of cell localization,it was observed that MRAK050699 was mainly located in the cytoplasm of lung tissue cells.KEGG pathway analysis showed that the top 10 up-regulated pathways were complement and coagulation cascade,starch and sucrose metabolism pathway,rheumatoid arthritis and so on.The first ten pathways of down-regulation are RAP1 signal pathway,endocytosis and so on.From the enrichment of GO,the high expression of Lnc RNAs-related target genes enriched 72 functional items in the cellular components.2.CCK8 results showed that the cell viability decreased significantly when the concentration of SiO₂dust was 40μg/cm².With the increase of SiO₂dust concentration,the expression level of TGF-βprotein increased gradually.RT-q PCR verified that the knockout efficiency of MRAK050699 was about 50%（F=8.659,P<0.05）.After co-culture,the morphological changes of RLE-6TN cells were observed under microscope.The cells in the normal group showed typical epithelioid characteristics,which were polygonal,oval and tight junction;the cells in the model group showed interstitial cell morphology,showing long spindle shape and spindle shape,and the intercellular gap widened;the knockout control group was similar to the model group,showing interstitial cell morphology.In the knockout group,only a small part of the cells changed from oval to spindle-shaped and spindle-shaped,and the cells were basically in a state of tight junction.The m RNA and protein expression levels of TGFbr1,p-Smad3,E-cadherin,N-cadherin,α-SMA,Vimentin,Snail,ZEB1,ZEB2,COLⅠ,Col III and FN in RLE-6TN cells were detected by PCR and WB.The results showed that compared with the normal group,the expression levels of TGFbr1,p-Smad3,N-cadherin,α-SMA,Vimentin,Snail,ZEB1,ZEB2,COLⅠ,Col III,FNm RNA and protein in the model group were increased,while the E-cadherin expression level was decreased.Compared with the knockout control group,the expression levels of TGFbr1,p-Smad3,N-cadherin,α-SMA,Vimentin,Snail,ZEB1,ZEB2,COLⅠ,Col III,FNm RNA and protein in the knockout group decreased,while the expression level of E-cadherin increased,but there was no significant difference between the model group and the knockout control group.Conclusion Long-term inhalation of SiO₂dust can cause silicosis fibrosis in rats.The lncRNA expression profile of silicosis model rats will change compared with normal rats.TGF-β/Smad signal pathway is an important fibrogenic pathway,and Tgfbr1 is the most important target gene in this pathway.Lnc RNAMRAK050699 can interfere with silicotic fibrosis by regulating the process of EMT.