Study On The Regulation Role Of LncRNA-ATB In Silicosis Fibrosis

Objective In vitro cell experiments were conducted to study the changes in the secretion of fibrosis factors and LncRNA-ATB by human embryonic lung fibroblasts induced by TGF-β1,and analyzed the correlation between LncRNA-ATB and the expression of these fibrotic factors;established a cell model of silicosis in vitro,and analyzed LncRNA-ATB in silicosis Regulation in fibrosis.Methods 1.Human embryonic lung fibroblasts(MRC-5)were cultured in vitro and stimulated with TGF-β1 at concentrations of 0,2.5,5.0,and 10.0 ng/m L for 24 h.The quantitative real time polymerase chain reaction(q RT-PCR)and western blot(WB)method to detect the long non-coding RNA activated by transforming growth factor β,collagen type Ⅰ(Col Ⅰ),collagen type Ⅲ(Col Ⅲ)and fibronectin(Fibronectin,FN)mRNA and protein expression level relatively.2.Liposome transfection conducted RNA interference against the si RNA of LncRNA-ATB,and the transfection effect was determined by q RT-PCR.MRC-5 cells were randomly divided into three groups:(1)ATB-si RNA group: transfected lnc RNA-ATB-si RNA;(2)NC-si RNA group: transfected si RNA nonsense sequence;(3)control group: normal cell culture.Phorbol ester(PMA)induces the differentiation of human monocyte THP-1 into macrophages.Macrophages were treated with free silica dust and co-cultured with the above three groups of cells.The MRC-5 cells in the lower chamber were collected after 24 hours,and the relative expression levels of FN,Col I,Col III mRNA and protein were detected by q RT-PCR and WB methods.Results 1.With the increase of TGF-β1 stimulation dose,cell proliferation activity was enhanced,and LncRNA-ATB,FN,Col I and Col III mRNA expression in the cells gradually increased,compared with the control group,difference was statistically significant(P<0.05).The result of correlation analysis showed that the mRNA level of LncRNA-ATB was positively correlated with the mRNA levels of FN(r=0.86),Col I(r=0.85)and Col III(r=0.82).After stimulation with different doses of TGF-β1,the expression levels of FN,Col I and Col III protein gradually increased,and the difference between the different dose groups and the blank group was statistically significant(P<0.05).2.Fluorescence in situ hybridization showed that LncRNA-ATB was expressed in the nucleus and cytoplasm of mrc-5.The expression of LncRNA-ATB was decreased in MRC-5cells transfected with ATB-si RNA,demonstrating the success of the LncRNA-ATB cell interference model.After co-culture,the mRNA expression levels of FN and Col I in the ATB-si RNA group were lower than those in the NC-si RNA group,and the difference was statistically significant(P <0.05).The expression level of Col III mRNA in ATB-si RNA group was not significantly different from that in NC-si RNA group(P>0.05).The protein relative expression levels of FN,Col I and Col III in ATB-si RNA group were lower than those in NC-si RNA group,and the difference was statistically significant(P <0.05).Conclusion TGF-β1 can stimulate fibroblast proliferation,LncRNA-ATB expression is up-regulated,FN,Col I and Col III mRNA and protein expression are increased,and LncRNA-ATB expression in cells is positively correlated with fibrotic factor expression.After LncRNA-ATB interference,the relative expression of FN and Col I decreased,and the expression of FN,Col I and Col III increased,suggesting that LncRNA-ATB plays a regulatory role in silicosis.