Selenium Modulates Chicken Skeletal Muscle Development Through MiR-365-3p Targeting SelT

Selenium（Se）is a human and animal essential trace element that maintains normal life activities in the body.Se deficiency cause a variety of organ tissue damage and nutritional metabolic diseases such as exudative qualities.Skeletal muscle is one of the major target tissues for Se deficiency.The biological function of Se in the body is achieved through selenoproteins.Selenoprotein T（SelT）,is abundantly expressed in multiple tissues and plays an important role in regulating oxidation,calcium homeostasis and neuroendocrine systems.At present,the study on the effect of SelT on skeletal muscle development is still unclear.MicroRNAs（miRNAs）are a class of conserved non-coding small RNAs that specifically bind to target genes and degrade mRNA or inhibit protein translation,ultimately modulating post-transcriptional expression of a target gene.MicroRNAs are involved in many biological processes and play an important role in cell proliferation,differentiation,development and apoptosis.miR-365-3p has a wide range of actions and participates in many pathological processes,but it has not been reported in skeletal muscle development.In this study,we replicated the Se-deficient chicken model,established miR-365-3p knockdown or overexpression and SelT knockdown chicken embryo model and chicken embryo myoblast model,examined the microstructure of skeletal muscle,muscle development-related factors,mitochondrial biosynthesis-related factors,and expression of calcium channel genes related to muscle development to indicate the biological function of SelT and miR-365-3p in chicken embryo skeletal muscle development.The test results are as follows:（1）Se deficiency lead to a decrease in the cross-sectional area of skeletal muscle fibers,and the gaps between muscle fibers become larger and less densely arranged,which significantly down-regulates the expression of SelT in skeletal muscle.The expression levels of MYF-5,MyoD,MyoG,MRF4,and MHC were significantly decreased,and the expression levels of MSTN and p21 were significantly increased.The expression of PGC-1α,NRF1,TFAM decreased,and the expression of STIM1 and TRPC1 decreased.The results showed that Se deficiency affected the development of skeletal muscles in terms of factors related to muscle development,mitochondrial biosynthesis,and calcium channel genes involved in muscle development.（2）Bioinformatics predictions show that SelT is the target gene of miR-365-3p,and the target gene is verified by luciferase detection system and Western blot technology,which confirmed that SelT is the target gene of miR-365-3p.（3）The miR-365-3p overexpression and knockdown model and the SelT knockdown model of chicken embryo myoblasts were constructed by using cell transfection technology;the overexpression of miR-365-3p and the knockdown of chicken embryos were established by the subembryonic injection route.Models and SelT knock down chicken embryo models.Morphological observations revealed that the miR-365-3p overexpression group and SelT knockdown group had reduced myoblast cell bodies,increased cell gaps,decreased intercellular connections,and even some cells died;miR-365-3p knockdown group.The poor state of myoblasts can be alleviated.In the chicken embryo model,when miR-365-3p was over-expressed,the cross-sectional area of skeletal muscle fibers of chick embryos was reduced,the gaps between muscle fibers were increased,loosely arranged and unevenly distributed;the skeletal muscle fibers of chicken embryos were reduced when SelT knocked down.The 365-3p overexpression group is similar.（4）MYF-5,MyoD,myogenin,MRF4,and MHC,which are beneficial to the development of skeletal muscle when overexpressing miR-365-3p and SelT knockdown,are down-regulated in chick embryo myoblast and chick embryo models,impeding skeletal muscle development.The expression of MSTN and p21 increased;knocking down miR-365-3p showed the opposite trend.The results showed that miR-365-3p regulate SelT to affect skeletal muscle development through muscle development related factors.（5）In the chick embryo myoblast and chick embryo models,over-expression of miR-365-3p,PGC-1α,NRF1,TFAM,decreased expression of SelT knockdown,intracellular mitochondrial number and ATP content decreased;Low miR-365-3p expression increased.The results indicate that miR-365-3p regulate SelT to affect skeletal muscle development through mitochondrial biosynthetic pathways.（6）In chicken embryo myoblast and chick embryo models,over-expression of miR-365-3p and SelT knockdown decreased the expression of STIM1 and TRPC1,and decreased cytoplasm,mitochondria,and endoplasmic reticulum calcium levels of myoblasts.The reverse test results were obtained after knocking down miR-365-3p.Therefore,the results indicate that miR-365-3p regulate SelT to affect skeletal muscle development through the Ca²⁺ pathway.