The Role And Mechanism Of Gli2 Regulation In Ductular Reaction

【Objective】Ductular reaction is a phenomenon of proliferation of bile duct cells induced by liver injury,which is related to various liver diseases such as liver cancer;research has shown that the Hedgehog（Hh）pathway is associated with ductular reaction,but there is a lack of direct research evidence and precise regulatory mechanisms to prove it.This study aims to investigate the role and mechanism of the transcription factor Gli2 in the bile duct response by knocking out the Hh pathway on bile duct cells.【Method】Obtaining CK19-Cre-ERT^-/-;m Tm G^flox/flox;Gli2^flox/floxgene mice through hybridization and mating,and then tamoxifen reagent was used to induce specific knockout of Gli2 on mouse bile duct cells.DDC induced ductular reaction.Detection of ductular reaction using HE,immunohistochemistry,and fluorescence quantitative PCR（q RT-PCR）.Detection of gene transcriptome expression changes in ductular reaction by RNA-seq.The protein expression levels of Hh,Yap1,and Notch pathways were detected using q RT-PCR and Western Blotting（WB）techniques.The regulation of Gli2 gene affects the analysis results in bioinformatics.Gli2 gene targets Yap1’s micro RNA and verifies candidate micro RNAs through q RT-PCR method.【Result】Tamoxifen treatment induces specific knockout of the Gli2 gene in bile duct cells.After inducing bile duct reaction using DDC,compared with the Gli2-WT group,the Gli2-KO group showed an increase in liver weight ratio,an increase in the number of Ep CAM and Sox9 positive bile duct cells,and an intensification of ductular reaction.The RNA-seq analysis results showed that in the Gli2-KO group,the metabolic activity of bile ducts in bile duct development and secretion increased,and the activity status of Yap1,Notch and other pathways related to bile duct development changed.The results of WB and q RT-PCR showed that the expression levels of transcription factors such as YAP1 and TEAD1in the YAP pathway,as well as the ligand Shh and transcription factor Gli1 in the Hh pathway,increased in the Gli2-KO group.36 micro RNAs regulated by Gli2 and targeting Yap1 were screened using bioinformatics analysis.After partial validation,it was found that the expression levels of mi R-30b-5p,mi R-30e-5p,and others were significantly downregulated.【Conclusion】Biliary duct cell specific knockout of Gli2 exacerbates ductular response.The increased activity of the Yap pathway and the compensatory increase of the Hh pathway transcription factor Gli1 are involved in the intensification of ductular response induced by Gli2 knockout.Mi R-30b-5p,mi R-30e-5p and other small RNAs may mediate Gli2 knockout induced increase in Yap activity.