The Mechanism Of MiR-128-1-5p Regulates Tight Junction Induced By Selenium Deficiency Via Targeting CADM1 In Broilers Vein Endothelial Cells

Selenium is one of the indispensable essential trace elements in the life process of plants and animals.Selenium can protect the cardiovascular system against oxidative stress,inflammation and other injuries.The lack of selenium or selenium content,not only affects the growth and development of animals,but also seriously restricts the animal’s health in the body.With the rapid development of life science and technology,there have been many studies on the biological functions of selenium-deficient mammals,but research on the gallus such as exudative diathesis remains to be in-depth.At the same time,the combination of new technologies such as cell molecular biology and genetic engineering has also attracted the attention of researchers,combining the use of non-coding RNAs such as miRNAs in the pathogenesis of selenium-deficiency disease in poultry.Therefore,it is particularly important to apply the research methods of miRNA to the research of animal diseases.As a non-coding short-chain RNA(20~25 nt),miRNA regulates the expression of posttranscriptional target gene mRNA through transcriptional repression.Although miRNAs have many functionally distinct family classifications,they all contain a “seed” at their 5’-end positions.High complementarity between seed and mRNA sequences is an important determinant of miRNA targeting.Among them,the miR-128 family is involved in the regulation of the vascular endothelial growth factor pathway,the generation of blood vessels,and the formation of epithelial and endothelial cell skeletons.Vein endothelial cells(VECs)are important barriers for mediating substance exchange in animals,and play an important role in blood’s colloidal osmotic pressure stability,nutrient transport,and regulation of vascular tone.The tight junctions formed between cells in VECs are important structural bases that mediate paracellular pathway transport.Whether selenium deficiency affects the clinical symptoms(exudative diathesis)of closely connected blood vessels participating in poultry has yet to be confirmed.In this study,miR-128-1-5p that was differentially expressed in vein tissue under selenium was screened from a number of candidate miRNAs based on the venous tissue subnets in the miRNA-regulated network of selenium-deficient broilers constructed in the our laboratory.Ultrastructural observation of venous tissues by transmission electron microscopy,combined with bioinformatics prediction,qRT-PCR and dual luciferase reporter gene methods,were used to analyze and screen potential miR-128-1-5p target genes.qRT-PCR and western blot techniques were used to verify the effect of knockdown/overexpression of miR-128-1-5p in VECs on tight junction function and cell bypass permeability;At the same time,flow cytometry was used to examine its effect on the cell cycle.The main research results are as follows:(1)Selenium deficiency leads to the damage of vein tissue and tight junction structure.The structural observation of broilers fed low-selenium diets showed that vein tissue was damaged,local inflammatory cells infiltrated,and the gaps between adjacent two intercellular spaces of VECs were enlarged and the connection structure was damaged.Selenium deficiency has been proven to cause vein tissue damage,cell disintegration,and impaired intercellular connectivity.(2)Determination of the miR-128-1-5p target gene.According to the results of the laboratory test,miR-128-1-5p,which was significantly differently expressed in vein tissue under selenium deficiency was screened out.Through bioinformatics prediction,qRT-PCR,western blot and dual luciferase reporter gene methods,the potential target genes were analyzed and screened,combined with KEGG pathway enrichment information and String protein interaction information to synthesize the exudative diathesis.The final selection and confirmation of the tight junction function-related gene CADM1 is a miR-128-1-5p targetgene,and there is a targeted relationship between mRNA and protein levels.(3)In vitro VECs test verified that miR-128-1-5p can regulate tight junction pathways.The knockdown/overexpression miR-128-1-5p model constructed in VECs successfully cultured in vitro was used to analyze the expression levels of key genes in the ZO-1 mediated tight junction pathway by targeting the CADM1 gene.Overexpression miR-128-1-5p indeed regulates the tight junction pathway and allows cells to contract.(4)In vivo exudative diathesis broiler model validation selenium regulation tight junction pathway.The expression levels of key genes in the tight junction pathway mediated by CADM1 and ZO-1 in the vein tissue of the selenium-deficient broiler model were analyzed by qRT-PCR and western blot techniques.Selenium deficiency leads to increased expression of miR-128-1-5p affects the expression of the target gene CADM1,which in turn regulates the tight junction pathway mediated by ZO-1,resulting in cell contraction and increased intercellular spacing.(5)miR-128-1-5p regulates changes in cell bypass permeability.VECs of knockdown/ overexpression miR-128-1-5p was marked with FITC-4-kDa and 70-kDa dextran.Overexpression of miR-128-1-5p is significantly increased paracellular permeability in either 4-kDa or 70-kDa dextran;knockdown miR-128-1-5p results decreased.Added the Fasudil,the paracellular permeability was decreased in VECs,returning to almost normal levels.HRP tracer reagents are consistent with dextran test results,indicating that impaired VECs barrier function is closely related to overexpression miR-128-1-5p.(6)miR-128-1-5p regulates cell cycle distribution.The cell cycle distribution caused by knockdown/overexpression of miR-128-1-5p was analyzed by flow cytometry.The results showed that overexpression of miR-128-1-5p can effectively increase the number of G1 phase cells.Knockdown miR-128-1-5p greatly increased the number of cells in S phase and G2/M phaseThis suggests that increased expression of miR-128-1-5p can cause G1 phase arrest of VECs.Fasudil inhibitors have a certain effect on cell cycle arrest.In summary,selenium deficiency can destroy tight junction structure of the intercellular barrier function.Through a series of analyses,miR-128-1-5p,which is highly expressed in the absence of selenium,can regulate the ZO-1 mediated tight junction pathway by targeting CADM1,leading to VECs contraction,tight junction structure changes,and cell bypass homeostasis.Impaired endothelial barrier function and induced G1 arrest in the cell cycle.This study fills the research gap of the pathogenic mechanism of broilers with selenium-deficient exudative diathesis,and provides a new research direction for enriching the research on the regulatory relationship between miRNA and tight junction pathway and understanding the function mechanism of cell barrier.