Rosiglitazone Deactivates Autophagy By Promoting MiR-29b Expression In Human Tenon’s Capsule Fibroblasts

Purpose:（1）To study the effects of rosiglitazone（RSG）on the expression of micro RNA-29b（miR-29b）and the levels of fibrosis and autophagy in human Tenon’s cystic fibroblasts（HTFs）,and compare the difference between RSG and transforming growth factor-β1（TGF-β₁）on miR-29b,so as to clarify the relationship between RSG and miR-29b.And explore the effect of miR-29b on fibrosis and autophagy during TGF-β₁-induced proliferation and activation of HTFs.（2）Analyze the genes and molecular pathways affected by RSG on the proliferation and activation of TGF-β1 induced by RNA-Sequencing,and the effects of RSG and miR-29b on the expression of key genes and signaling pathways were verified.And find the interaction between fibrosis and autophagy in the proliferation and activation process of HTFs induced by RSG,miR-29b and TGF-β₁,to further analyze the mechanism of RSG,and to provide a more adequate theoretical basis for the clinical application of RSG.Methods:（1）Different TGF-β₁concentration gradients and time gradients were set,and the expression of type I collagen（type I collagen,ColI）and connective tissue growth factor（connective tissue growth factor,CTGF）after TGF-β₁activated HTFs was detected by Westerm-Blot（WB）to determine the optimal concentration and duration of TGF-β₁.（2）Different RSG treatment time gradients were set to determine the effect of RSG alone on miR-29b expression in HTFs by q-PCR,and to compare the effects of RSG and TGF-β₁on miR-29b expression in HTFs.（3）TGF-β₁was used to induce activation of HTFs to establish a cytological model of filter channel scarification,while using adenovirus to construct miR-29b overexpression/inhibition infection system.The effects of RSG and miR-29b on the expression levels of fibrosis related to of HTFs（includingα-SMA,Col 1,CTGF,SP1）and autophagy（including P62,light chain 3I（light chain 3I,LC3I）and LC3II）were tested and compared by q-PCR and WB.（4）The differential expression of gene level and transcript level in HTFs were analyzed by RNA-Sequencing,TGF-β₁induced activation and treated with DMSO/RSG,select differentially expressed genes between samples and groups,dig and analyze the data,cluster differentially expressed genes,GO function significance enrichment analysis,Pathway significance enrichment analysis,etc.（5）TGF-β₁was used to induce HTFs activation,a cytological model of filter channel scarring was established,and the expression of m-TOR pathway,PI3K-Akt pathway,AMPKαpathway and P53 pathway related genes and signaling molecules was detected by WB and q-PCR techniques.（6）Use Excel software to collect and collate the data and establish a database.Results were statistically analyzed and graphed by Image J and Graph Pad Prism 9.Means and standard deviation were calculated for each data,comparing data between two groups using independent sample t-test and between multi-groups by one-way analysis of variance（one-way ANOVA）with statistical significance expressed as P<0.05.Results:（1）TGF-β₁can effectively promote the extracellular matrix expression of HTFs and induce cell fibrosis,and the effect increases with the increase of TGF-β₁concentration and the treatment time;However,when the TGF-β₁concentration reaches 20 ng/m L or the treatment time reaches 12h,the effect gradually decreases.TGF-β₁downregulated the expression of miR-29b in the cells,which was positively correlated with the concentration.（2）When RSG treated HTFs 6h,COL I and CTGF protein expression began to decrease significantly,reaching a peak at 8h,after which the inhibitory effect of RSG on fibrosis was weakened over time.Meanwhile,RSG upregulated miR-29b expression in the cells,which was first enhanced with the increase of the intervention duration and then gradually weakened.（3）Upregulation of miR-29b expression in HTFs inhibited the m RNA expression of P62,LC3B,α-SMA and COL I,and promoted the m RNA expression of CTGF and SP1.Meanwhile,upregulated expression of miR-29b in cells promoted the degradation of P62 and the conversion of LC3I to LC3II,and inhibited the expression ofαSMA,COLI,CTGF and SP1 proteins,and the number of autophagosomes in cells was increased significantly.Moreover,miR-29b negatively regulates the expression of target genes PI3Kp85αm RNA and proteins.（4）By cross analysis of differentially expressed genes,we found that there were 10 HTFs downregulated after TGF-β₁intervention and upregulated by RSG treatment,13 upregulated after TGF-β₁intervention and downregulated by RSG treatment,among which DUSP 6 and MAP3K5 were closely related to MAPK signaling pathway.Cross-KEGG pathway analysis of genes differentially expressed in HTFs after TGF-β₁intervention and RSG treatment found that genes upregulated after TGF-β₁intervention and downregulated after RSG treatment were associated with 24 pathways related to inflammatory immune response,signaling pathway related to tumor aging,VEGF signaling pathway,PI3K-Akt signaling pathway,JAK-STAT signaling pathway and MAPK signaling pathway.Genes downregulated after TGF-β₁intervention and upregulated after RSG treatment were only associated with one pathway,the MAPK signaling pathway.Upon validation of the results of Rna-seq analysis,the m RNA and protein expression levels of DUSP 6 and ASK1 were significantly increased in HTFs after RSG intervention and upregulation of miR-29b;the m RNA and protein expression levels of ERK1/2 were significantly decreased,but the m RNA and protein expression levels of P38 were significantly increased.（5）RSG intervention and upregulation of miR-29b can inhibit the expression of PI3Kp85α,Akt,RSK2 and mTOR in HTFs;meanwhile,promote the expression of TSC1/2,ULK1,PI3KC3,Beclin1,FIP200,Atg13,Atg14,AMPKαand P53 in HTFs.Conclusions:（1）TGF-β₁can promote the extracellular matrix expression of HTFs and induce cell fibrosis,and this effect is concentration-dependent;RSG can inhibit the fibrosis of HTFs cells,and this effect is enhanced with the increase of intervention time.（2）TGF-β₁down-regulated miR-29b expression in cells in a concentration-dependent manner,and RSG up-regulated miR-29b expression in cells.（3）RSG can upregulate miR-29b expression in HTFs cells and activate cellular autophagy to block TGF-β₁-induced cell activation of HTFs,thereby inhibiting cell fibrosis of HTFs.（4）RSG regulates the expression of PI3Kp85α,DUSP6 and MAP3K5 genes by upregulating the level of miR-29b in HTFs,activates the P38 MAPK pathway,P53 pathway and AMPK pathway while inhibiting the expression of PI3K-Akt pathway and ERK pathway,and inhibited the activity of mTOR under the TSC1/2 protein complex,and activated Beclin1-Atg14 pathway to induce autophagy,thus inhibiting the fibrosis of HTFs.