The Role Of LAMB3 On Myofibrogenic Fibrosis Of Endometrial Stromal Cells And The Regulation Mechanism Of Intrauterine Adhesions

Aims and significance To identify the differential gene laminin beta-subunitβ3(LAMB3)in severe intrauterine adhesions by transcriptome sequencing,to investigate the effect of LAMB3 in regulating myofibrogenic transformation of endometrial Stromal cells,and to explore the mechanism of LAMB3 in intrauterine adhesions by bioinformatics analysis of downstream regulatory pathways.Methods In the first part,differentially expressed genes are identified in endometrial tissues of the intrauterine adhesion group and the control group by transcriptome sequencing.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses are applied to find significantly differentially expressed pathways.The reliability of transcriptome sequencing is verified by real-time fluorescent quantitative PCR(qRT-PCR).Endometrial expressions of the Messenger Ribonucleic Acid(mRNA)and protein of the significantly differential gene LAMB3 are validated in the intrauterine adhesion group and the control group using qRT-PCR and immunohistochemistry.In the second part,stable transfected cell lines of endometrial stromal cells(ESC)silencing and overexpressing LAMB3 are constructed.The effect of LAMB3 on myofibroblast phenotypic transformation of endometrial stromal cells is elucidated by examining the changes of myofibroblast biomarker expression in endometrial stromal cells.The localization of intracellular fibrous actin(F-actin)by phalloidin is used to investigate the effect of LAMB3 on ESC cytoskeletal remodeling.In the third part,the effect of LAMB3 on endometrial fibrosis,embryo implantation and live births of rats with intrauterine adhesions is further validated by constructing an animal model of intrauterine adhesions through vivo animal experiments.In the fourth part,the pathways of silenced LAMB3 and overexpressed LAMB3 and the reversal of myofibrosis are verified in ESC cells by the activator of the RhoA/ROCK1/MYL9 pathway,oleoyl-L-α-lysophosphatidic acid and the inhibitor Y-27632 in both directions.In an animal model of rat intrauterine adhesion,the effect of Y-27632 on endometrial fibrosis,embryo implantation and live births of rats with intrauterine adhesions is verified.Results1.399 differentially expressed genes are identified by transcriptome sequencing in endometrial tissues of the intrauterine adhesion and control groups.Enrichment scores of GO enrichment analysis in descending order are metalloendopeptidase activity,voltage-gated potassium channel activity,muscle contraction and extracellular region.Pathways analysed for significant differences by KEGG enrichment analysis include vascular smooth muscle contraction,focal adhesion,and tumor necrosis factor(TNF)signalling pathways.2.The reliability of transcriptome sequencing is verified by qRT-PCR.Transcriptome sequencing,qRT-PCR and immunohistochemistry results all show higher mRNA and protein expression of LAMB3 in the intrauterine adhesion group than in the control group,identifying LAMB3 as the target gene for the next study.3.By detecting the changes of myofibrogenic biomarker expression in ESC,LAMB3 is elucidated to promote myofibroblast phenotype transformation in endometrial stromal cells.4.Activation and silencing of LAMB3 promotes ESC cytoskeleton remodeling,resulting in altered cell morphology and distribution of actin filaments.5.Vivo animal experiments further demonstrate that interference with LAMB3 alleviates endometrial fibrosis of rats with intrauterine adhesions,and silencing LAMB3 improves the number of embryo implantations and live births and increases fertility of rats with intrauterine adhesions.6.LAMB3 regulates myofibrogenic transformation of endometrial mesenchymal cells via the RhoA/ROCK1/MYL9 pathway.7.Y-27632 significantly improves endometrial fibrosis and fertility of rats with intrauterine adhesions.Conclusion This study confirms by clinical tissue samples,in vitro cellular molecular experiments and in vivo animal experiments that LAMB3 regulates myofibrogenic transformation of endometrial mesenchymal cells through the RhoA/ROCK1/MYL9 pathway,which ultimately affects endometrial fibrosis,embryo implantation and live birth number.LAMB3 can be considered as a heterogeneous biological molecule of intrauterine adhesion to involve in the endometrial fibrosis process and provides a new target for gene therapy and drug intervention by interfering with RhoA/ROCK1/MYL9 pathway regulation.