| Objectives Aiming at the regulation and function of silicotic macrophages,this research explore molecular mechanism of lnc RNA NEAT1 regulating macrophage autophagy,study upstream regulatory network of macrophage autophagy,clarify the role and mechanism of lnc RNA NEAT1 regulating silicotic fibrosis through macrophage autophagy.Methods 1 Modeling: Silicosis model and control model were established,which were prepared for single cell transcriptome detection.Silicosis intervention models(non intervention,si Ctrl,si Beclin1,si Ctrl+Lenti-Ctrl,silnc RNA NEAT1+Lenti-Ctrl,silnc RNA NEAT1+Lenti-E2F1,silnc RNA NEAT1+Lenti-SKP2,silnc RNA NEAT1+si Ctrl,silnc RNA NEAT1+si Beclin1)and control model were established,which were used for signal pathway verification.2 Cell intervention: Rapamycin,3-MA,CQ,CS were used for cell drug treatment.Silencing cells were treated with si Ctrl,si Beclin1,si E2F1,si SKP2,silnc RNA NEAT1,mi R-205-5p inhibitor.Overexpression cells were treated with oe Ctrl,oe Beclin1,oe E2F1,oe SKP2,oelnc RNA NEAT1,mi R-205-5p mimics.3 Index detection:RNA FISH was used to detect the localization of lnc RNA NEAT1 in cells.RNA pulldown was used to detect the interaction of lnc RNA NEAT1 with E2F1 and mi R-205-5p.RIP was used to detect the binding of lnc RNA NEAT1 to E2F1 and mi R-205-5p.IP was used to detect the interaction between SKP2 and Beclin1.Dual luciferase reporter genes were used to verify the activity of SKP2 promoter regulated by E2F1,the binding of lnc RNA NEAT1 to mi R-205-5p and mi R-205-5p to E2F1.HE staining,Masson staining were used to detect pulmonary fibrosis.Immunohistochemistry was used to detect E2F1,SKP2,Beclin1,LC3.Transcription levels of NEAT1,mi R-17-5p,mi R-20-5p,mi R-93-5p,mi R-106-5p,mi R-205-5p,mi R-519-3p,E2F1,SKP2,Beclin1,Col1a1,Col3a1 were detected by q RT-PCR.Protein levels of E2F1,SKP2,Beclin1,LC3,ATG5,Col1a1,Col3a1 were detected by WB.Autophagy was observed by double luciferase reporter assay.4 Statistical analysis: t-test,variance analysis,Pearson correlation analysis.Results 1 Through single-cell transcriptome analysis,NEAT1 was closely related to macrophage apoptosis in silicosis model.2 Mouse model demonstrated the relationship between lnc RNA NEAT1 and silicosis fibrosis:(1)Histological study showed that lnc RNA NEAT1 was highly expressed in lung tissue of silicosis mice,while fibrosis was obvious;(2)Relationship between silicosis fibrosis and macrophage autophagy: After injection of si Beclin1,Col1a1 and Col3a1 were highly expressed in lung tissue of silicosis mice(P<0.01).LC3-I,LC3-II,ATG5 were highly expressed in Beclin1 overexpressed MH-S cells,while LC3-II/LC3-I increased.Silence of Beclin1 was on the contrary(P<0.01);(3)Relationship between Beclin1 and upstream regulator SKP2,transcription factor E2F1,mi R-205-5p: After si SKP2 transfection into MH-S cells,Beclin1 was highly expressed,while stability of Beclin1 increased.Which was further confirmed that SKP2 connected Beclin1 through K48 polyubiquitination.After silencing E2F1 in MH-S cells,SKP2 showed low expression.Overexpression of E2F1 had the opposite effect(P<0.01).E2F1 was positively correlated with SKP2(r=0.7734,P=0.0082).In MH-S cells,mi R-205-5p mimics could reduce the expression of E2F1,while mi R-205-5p inhibitors had the opposite effect(P<0.01).It was confirmed that mi R-205-5p could regulate the expression of E2F1;(4)Lnc RNA NEAT1/mi R-205-5p/E2F1 axis: After silnc RNA NEAT1 transfection into MH-S cells,E2F1 showed low expression.Compared with silnc RNA NEAT1 group,E2F1 was highly expressed after silnc RNA NEAT1+mi R-205-5p inhibitor transfection(P<0.01);(5)Lnc RNA NEAT1/E2F1/SKP2 axis: Lnc RNA NEAT1 was positively correlated with SKP2(r=0.7731,P=0.0084).After silnc RNA NEAT1 transfection into MH-S cells,SKP2 showed low expression.After oelnc RNA NEAT1 transfection,the opposite was true(P<0.01).Compared with oelnc RNA NEAT1 group,SKP2 showed low expression after oelnc RNA NEAT1+si E2F1 transfection(P<0.01).3 In vitro cytological experiments demonstrated the relationship between lnc RNA NEAT1/E2F1/SKP2/Beclin1 and macrophage autophagy: After silnc RNA NEAT1 transfection into MH-S cells,Beclin1 was highly expressed,while E2F1 and SKP2 were lowly expressed.Oelnc RNA NEAT1 transfection was on the contrary(P<0.01).4 In vivo experiments in mice further demonstrated the relationship between lnc RNA NEAT1/E2F1/SKP2/Beclin1 and silicosis fibrosis: Compared with Lenti-Ctrl+si Ctrl group,transcription level of mi R-205-5p increased,E2F1,SKP2,Col1a1,Col3a1 were lowly expressed,while Beclin1 and LC3 were highly expressed,pulmonary fibrosis decreased in Lenti-Ctrl+silnc RNA NEAT1 group.Compared with Lenti-Ctrl+silnc RNA NEAT1 group,E2F1 was highly expressed in Lenti-E2F1+silnc RNA NEAT1 group,while SKP2,Col1a1,Col3a1 were highly expressed in Lenti-SKP2+silnc RNA NEAT1 group,but Beclin1 and LC3 were lowly expressed,pulmonary fibrosis increased(P<0.01).Compared with si Ctrl+silnc RNA NEAT1 group,Beclin1 and LC3 were lowly expressed,while Col1a1 and Col3a1 showed highly expression,pulmonary fibrosis increased in si Beclin1+silnc RNA NEAT1 group(P<0.01).Conclusions 1 NEAT1 in macrophages is differentially expressed in single cellomics.2Lnc RNA NEAT1 can be used as ce RNA to relieve the targeted inhibition of transcription factor E2F1 by mi R-205-5p.3 Lnc RNA NEAT1 can recruit E2F1 to SKP2 promoter and promote its expression.4 Upstream regulator SKP2 connects Beclin1 through K48 polyubiquitination and promotes Beclin1 degradation.5 Autophagy effector protein Beclin1 can promote macrophage autophagy.Inhibition of Beclin1 can promote pulmonary fibrosis.6 Lnc RNA NEAT1 inhibits macrophage autophagy through E2F1/SKP2/Beclin1 axis and promotes silicosis fibrosis.Figure 87;Table 38;Reference 243... |
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