Selenium Deficiency Induce Chondrocyte Apoptosis Through Gga-miR-138-5p Targeting SelM-activated Mitochondrial Pathway

Many essential trace elements play important roles in human and animal life,and selenium(Se)is one of them.Se is closely related to the human and animal health.Se deficiency can lead to the occurrence of Keshan disease in human and exudative diathesis,white muscle disease and other pathological changes in animals.Se also plays important roles in the normal functions of cartilage.Low Se content in the organism may directly influence the occurrence of the somatic motor disease,such as Kaschin-Beck disease(KBD)and osteoarthritis(OA).In the organism,Se usually exerts its regulation function in the form of Selenoproteins.Until now,more than 30 gene sequences of different animals have been detected.Selenoprotein M(SelM)is one of the selenoprotein thioredoxin reductase family members(Txnrds),which can be expressed in various tissues of different animals.For the time being,SelM has been conformed to have antioxidative functions in the chicken,but the mechanisms of its protective function against apoptosis in the chicken cartilage tissue were still unclear.To further understanding the function of SelM in the cartilage tissue,in the current study we established Se-deficiency chicken model,detected the mRNA expression levels of 24 selenoproteins,the protein expression levels of SelM and the oxidative stress index(H2O2、CAT、SOD and MDA),and established the over-expression(Mimic group)and knockdown(inhibitor group)models of gga-miR-138-5p,which targets SelM,by transfecting the Mimic and inhibitor of gga-miR-138-5p into the chicken tibial cartilage cells cultured in vitro.By using flow cytometry,kits,qPCR and Western blot,the rate of apoptosis,oxidative stress indexes(H2O2、CAT、SOD and MDA)and the mRNA and protein levels of apoptosis-related genes(BCL-2、Caspase-3、Caspase-9、BAX and BAK)were examined and the mechanisms of SelM on protecting the chicken cartilage tissue against the abnormal apoptosis induced by oxidative injury were elucidated.The results are as follows:(1)The mRNA expressions of 24 selenoprotein in the C group and L group were detected and carried on comparative analysis,and the data showed that the expression of SelM in the chicken tibia cartilage was the highest and greatly affected by the changes of Se content in diet;the protein expression of SelM in L group was 75%of that in C group.The results show that selenium deficiency can significantly reduce the expression of SelM.(2)The establishment of chicken primary chondrocytes in vitro culture model based on the construction of miR-138-5p overexpression and knockdown model,the results show the optimal transfection concentration of miR-138-5p Mimic was 50nM,and the overexpression efficiency was 22.5 times.The mRNA expression of SelM gene was only 25%and the protein expression was 69%compared with C group.The optimal transfection concentration of miR-138-5p Inhibitor was 100nM,knocking down efficiency was 0.51 times,while SelM gene expression was 4.47 times that of C group,and the protein expression was 1.66 times of that of C group.Additionally,the results of dual luciferase reporter assay system showed that SelM is the target gene of gga-miR-138-5p.The results demonstrate that miR-138-5p targets SelM and regulates its expression.(3)Compared with the C group,the levels of apoptosis,H2O2 and MDA were increased and the activities of CAT and SOD were decreased in the chicken tibial cartilage of L group;the mRNA and protein levels of Caspase-3,Caspase-9,BAX and BAK showed an upward trend,that of BCL-2 decreased significantly;miR-138-5p expression increased;cell apoptosis increased significantly(P<0.05).In vitro test results show that,compared with C group,the contents of H2O2 and MDA were significantly increased and the activities of CAT and SOD were significantly decreased,and the mRNA and protein levels of Caspase-3,Caspase-9,BAX and BAK showed an upward trend and that of BCL-2 showed an downward trend in the Mimic group.The contents of H2O2 and MDA showed an downward trend and the activities of CAT and SOD were significantly increased,and the mRNA and protein levels of Caspase-3,Caspase-9,BAX and BAK were lower and that of BCL-2 was higher in the cartilage cells of inhibitor group compared with C group.These results suggest that selenium deficiency can upregulate the expression of miR-138-5p,down-regulate the expression of SelM,and induce oxidative stress in chondrocytes,and induce apoptosis of chondrocytes through mitochondrial pathway.In conclusion,Se deficiency can reduce the expression of 24 selenoproteins in cartilage tissue,upregulate the expression of miR-138-5p,and miR-138-5p induced oxidative stress in chondrocytes by targeting SelM,and induced chondrocyte apoptosis through mitochondrial pathway.