The Role Of MiR-215-5p Targeting CTCF Pathway In Myocardial Dysplasia Induced By Selenium Deficiency In Chicken

Selenium(Se)is a trace element essential for the growth and development of myocardium in animals and humans.Se deficiency can induce a variety of myocardial diseases,such as human Keshan disease,myocardial degenerative white muscle disease,myocardial necrosis injury and dilated cardiomyopathy.Myocardial development can be regulated by a variety of transcription factors and microRNAs.The results of pre-application micro RNA showed that the expression of miR-215-5p in Sedeficient myocardium of chicken increased by 152%.The target gene website prediction results indicated that CTCF may be one of the miR-215-5p target genes.miR-215-5p is widely used and involved in many pathological processes.However,the specific mechanism of miR-215-5p during Se-deficient myocardial development remains unknown.Further,through using quantitative real-time fluorescent quantitative PCR(q RT-PCR),Western blotting(WB)and dual luciferase reporter gene systems(LRGS)to verify whether CTCF is a target gene of miR-215-5p,and it reveals its specific biological functions of cardiomyocytes and myocardium in chicken.Based on the establishment of Se-deficient broiler model and the overexpression/knockdown of miR-215-5p in cardiomyocytes model,we observed the morphological changes of Se-deficient myocardial tissue and cardiomyocytes model,and identified the target gene of miR-215-5p.At the same time,the m RNA and protein levels of myocardial transcription factors,myocardial development-related factors,mitochondrial biosynthesis-related factors were detected,and the number of mitochondria and ATP levels in cardiomyocytes were detected.The results show as follows:1.The connection of myocardial fiber and the bundle of myocardial fiber decreased obviously,and the gap of myocardial fiber was increased in Se-deficient myocardial tissue.We further analyzed the changes of myocardial transcription factors,mitochondrial biosynthesis-related genes,myocardial development-related factors at the m RNA and protein levels in Se-deficient myocardium.Combined with the observation of myocardial morphology,the results indicated that Se deficiency can cause the decrease of myocardial transcription factors,mitochondrial biosynthesis disorder,the down-regulation of myocardial development related factors,and the content of ATP in Se-deficient myocardium,which would affect the normal development of myocardium of chicken.2.Using bioinformatics prediction website Target Scan and Mi RDB to predict for miR-215-5p target genes,on the basis of chicken cardiomyocyte model with overexpression and knockdown of miR-215-5p,q RT-PCR,WB and LRGS were used to verify that CTCF is the target gene of miR-215-5p.3.Construction of cardiomyocyte miR-215-5p overexpression/knockdown model by cell transfection technique.Morphological observation showed that overexpression of miR-215-5p caused myocardial cell gap to become larger,and the intercellular junctions were significantly reduced,at the same time myocardial fibers and myocardial fiber bundles were disintegrated,and the intercellular pseudopodia was not interwoven into a network.The poor growth of cardiomyocytes in the miR-215-5p knockdown group could be alleviated,and the cardiomyocytes were tightly bound to form radial or concentric clusters of cells.4.The m RNA and protein expression of transcription-related factors(Hey2,Irx4,Mef2 d and Snail1,etc),myocardial development-related genes(Gata4,Nkx2-5,Tnn T2,myogenin,Wnt11,etc.)were downgraded.The opposite trend was observed in the miR-215-5p knockdown group.The results indicated that miR-215-5p targeted CTCF to regulate myocardial development-related genes,thereby affecting myocardial developmental of chicken.5.In the miR-215-5p overexpressing chicken cardiomyocyte model,the m RNA and protein expression levels of mitochondrial biosynthesis-related genes(PGC-1α,TFAM,TFB2 M,NRF1,NRF2 and ND2)were reduced.The number of mitochondria and ATP content in cardiomyocytes were decreased;however,the expression of m RNA and protein of the above genes was increased in the miR-215-5p knockdown group.The results indicated that miR-215-5p targeted CTCF to affect myocardial development through mitochondrial biosynthesis pathway.In combination with experiments in vitro and in vivo,Se deficiency could induce the increase of miR-215-5p expression,which could result in the disorder of myocardial transcription genes,the imbalance of mitochondrial biosynthesis,and decreased expression of genes related to myocardial development.In addition,miR-215-5p may target CTCF to regulate myocardial development-related factors via a non-canonical Wnt signaling pathway and induce mitochondrial dysfunction through the PGC-1α-TFAM-NRF1/2 pathway in the heart of Se-deficient chickens.Our results not only demonstrated that Se deficiency inhibited myocardial development by directly targeting the miR-215-5p/CTCF pathway,and miR-215-5p inhibitor can promote normal myocardial development through non-canonical Wnt signaling pathways.Our results reveal a regulatory mechanism: miR-215-5p-targeted CTCF pathway induced by Se-deficiency could regulate myocardial development,which provides a new way to study the mechanism of Se deficiency-induced myocardial dysplasia.