Role Of Golm1 And Autophagy In Pulmonary Fibrosi

Part1:The role of Golml in the development of pulmonary fibrosis and the potential molecule mechanism investigationBackground:Fibrotic interstitial lung diseases(pulmonary fibrosis),including idiopathic pulmonary fibrosis(IPF),are a group of unknown etiology and pathogenesis,which seriously affect the patients’survival and life quality and impose a heavy burden on families and society.IPF is a chronic progressive and fatal lung disease.Various factors contribute to the development of IPF,such as abnormal proliferation of fibroblasts,abnormal expression of myofibroblasts,damage to the lung epithelium or endothelium,abnormal tissue repair,dysregulation of wound healing pathways,aberrantly production and accumulation of extracellular matrix(ECM).In the current research,we aimed to explore the role Golgi membrane protein 1(Golml)in pulmonary fibrosis and potential molecular mechanism of Golml in promoting fibrosis.Methods:We first analyzed the expression of Golml in IPF patients as well as control lung tissues and serum through the GEO public database.Second,we collected sera as well as lung tissues from clinical IPF patients and donor controls.Golml expression was detected by ELISA,qRT-PCR,Western Blot and immunohistochemistry,respectively.Subsequently,we monitored Golml expression in various fibroblasts stimulated by TGF-β1 as well as bleomycin-induced mice fibrosis models.To further explore the role of an increased Golml in fibrosis,we constructed cell lines with stable silencing and stable overexpression of Golml using lentivirus,and detected fibrosis-related proteins by Western Blot.Besides,Cells’ migration and proliferation ability were also detected using Transwell and CCK8,respectively.Additionally,we constructed systemic Golml knock-in and systemic knockout mice,and examined fibrosis-related biomarkers using bleomycin-induced fibrosis mouse model to investigate the role of Golml in fibrosis.To further investigate the mechanism of Golml-promoted fibrosis,we performed the whole transcriptome sequencing(RNA-Seq)over Golml-silenced and overexpressed cells,and screened that lncRNA Neatl downstream Golml could be positively regulated by Golml.Further functional experiments of Neatl on fibrosis were performed on fibroblasts with Golml silencing and overexpression cells using Western Blot,Transwell and CCK8.Finally,we determined that Golml could inhibit expression of Klf4 which can binding to the promotor of Neatl results in an inhibited Neatl transcriptional expression through GSEA database,ChIP and other experiments.Results:We first identified Golgi membrane protein 1(Golml)significantly elevated in lung tissue and blood in IPF patients through the GEO public database.Subsequent validation in lung tissue collected from clinical IPF lung transplant patients,as well as in patients’ serum specimens also revealed significantly elevated Golml in IPF patients in comparison with the control.Next,we knock down and overexpress Golml,respectively using human and mouse fibroblasts.A series of functional experiments were performed to validate that Golml not only has a pro-fibrotic effect,but also synergizes with TGF-β1 during fibrogenesis to further aggravate the development of fibrosis.Additionally,we constructed pulmonary fibrosis mouse model by tracheal injection of bleomycin using systemic knock-in Golml and systemic knockout Golml mice.The results showed that systemic knockout Golml mice developed a minor fibrosis,while systemic knock-in Golml mice had more seriously formation of fibrosis,accompanied to lower body weight and survival rate in comparison to the wildtype.By analyzing and validating the whole-transcriptome gene sequencing(RNA-Seq)on the constructed Golml stable knockdown and overexpression cell lines,we screened that lncRNA Neatl can be positively regulated by Golml.Golml promotes the expression of Neatl and thus the development of fibrosis.Furthermore,analysis of the promoter of Neatl has indicated that Klf4 could be a regulator.expression of Golml and Klf4 are in negative correlation analyzed by GSEA.Further experiments conducted using Golm1 silenced and overexpressed cells also demonstrates that Golml negatively regulates Klf4.Finally,to elucidate the mechanism of Klf4 in negative regulation of Neatl,ChIP was performed and the results have indicated that Klf4 can bind to the promotor of Neatl thus transcriptionally inhibits Neat1 expression.Conclusion:For the first time,this study explains for the role of Golml in pulmonary fibrosis.Golml promotes the development of fibrosis by repressing the expression of Klf4 and thus promoting the transcription of Neat1.The abnormal expression of Golm1 may shed a mechanism insight for the future treatment of pulmonary fibrosis patients.Part 2:Dynamic Observation of Autophagy and Transcriptome Profiles in a Mouse Model of Bleomycin-Induced Pulmonary FibrosisBackground:Fibrotic interstitial lung diseases(pulmonary fibrosis),including idiopathic pulmonary fibrosis(IPF),are a group of unknown etiology and pathogenesis,which seriously affect the patients’ survival and life quality and impose a heavy burden on families and society.Recently,autophagy in pulmonary diseases has aroused a great concern.However,the role of autophagy in pulmonary fibrosis is controversial.Our study aimed to explore the role of autophagy in various stages of fibrosis and to understand its changes in each stage.Methods:In the current research,we dynamically observed a bleomycin-induced pulmonary fibrosis mouse model after 3,7,14,21,and 28 days and investigated the expression of autophagy markers.Lung tissues were collected and autophagy marker LC3B and SQSTM1 protein were detected by Western Blot.Autophagic vesicles were observed under an electron microscopy and immunofluorescence were used for evaluating the fluorescent spots of LC3B.Subsequently,we extracted total RNA from lung tissues of mice at different days post-bleomycin and whole transcriptome gene sequencing(RNA-Seq)was performed for investigation of differentially expressed genes.Short-time sequence expression probe(STEM)analysis was performed to explore the expression of genes associated with different stages and time points of fibrosis.The functions and involved signaling pathways of differentially expressed genes at each time points post-bleomycin were analyzed by gene ontology function(GO)as well as KEGG signaling pathway.Meanwhile,autophagy-related genes in the transcriptome were also analyzed and compared by heat map.Additionally,we also validated them by Western Blot.Finally,autophagy-related genes’ expression in specific cells were analyzed from a publicly available database of three human and one animal study of pulmonary fibrosis using single-cell sequencing technology.Results:We found no significant changes of autophagy markers in the lung tissue of mice with pulmonary fibrosis at the specified time points.RNA-Seq has revealed many differentially expressed genes.By STEM analysis of consistently up-or down-regulated genes as well as functions and pathways they involved,we did not find any autophagy-related genes that play a sustained role in fibrosis development.In addition,GO analysis have showed that the persistently upregulated gene profiles were mainly related to fibrosis synthesis,extracellular matrix and inflammation,while the enriched signaling pathways were mainly implicated in PI3K-AKT,ECM-receptor interactions and cell adhesion signaling pathway.For the consistently down-regulated gene expression profile,GO functions were mainly involved in myofibrillar organization,muscle contraction and myofiber development.The enriched KEGG signaling pathways were cAMP,cGMP-PKG,calcium signaling pathway and myocardial contraction-related pathways.Moreover,expression of autophagy-related genes in specific cells from a publicly available database of three human and one animal study of pulmonary fibrosis using single-cell sequencing technology have also shown no critical role of autophagy in the pathogenesis of pulmonary fibrosis.Conclusion:Autophagy may not critically and consistently change during the development of pulmonary fibrosis at different stages post-bleomycin in a mouse model.These continually up-or downregulated profiles,including gene profiles,and the corresponding functions and pathways may provide mechanistic insights into IPF therapy.