MiR-33-3p Involved In Selenium Deficiency-induced Chicken Kidney Tissue Apoptosis By Targeting ADAM10 And Regulating ?-catenin Pathway

Selenium is a key trace element that plays an important role in biology.Dietary selenium deficiency can cause a series of typical clinical and pathological changes in various chicken organs.And the kidney has higher selenium level compared with other tissues,and the kidney is one of the target organs of selenium deficiency.Selenium deficiency induces kidney function decline,oxidative stress and kidney fibrosis,and also affects cell cycle progression and cause apoptosis.However,selenium supplementation relieves these symptoms.Studies have shown that miRNAs also plays an important role in the development of kidney disease.MiR-33 is a highly conserved miRNA family,which plays an important role in cell cycle and apoptosis,however,the role of miR-33 in renal cell apoptosis in selenium-deficient broilers remains unclear.In order to investigate the role of miR-33-3p and its target genes in the cell cycle and apoptosis of seleniumdeficient kidney cells,the present study was carried out by duplicating the model of selenium deficient chicken,and using ultrastructural observation,real-time fluorescence quantitative PCR,luciferase double reporter gene,AO-EB and so on,we detected the mRNA expression of ?-catenin-associated genes(E-cadherin,?-catenin,cyclinD1,TCF,c-myc,survivin)and apoptosisrelated genes(Bcl-2,Bax,Bak,Caspase3)as well as ADAM10 and miR-33-3p in seleniumdeficient kidneys.On the basis of the establishment of model with miR-33-3p overexpression and knockdown in CEK cells,we predicted and validated the target gene of miR-33-3p was ADAM10,and detected the expression of ?-catenin pathway related genes and apoptosis-related genes in the kidney tissue by knockdown and overexpression of miR-33-3p in CEK cells cultured in vitro.The results showed that:(1)On the basis of the establishment of the CEK cells in vitro,the overexpression and knockdown model of miR-33-3p was successfully constructed,and the overexpression efficiency was 46 times and the knockdown efficiency was 0.45 times compared with control group.The target gene of miR-33-3p was predicted by bioinformatics prediction software TargetScan and miRDB,and miR-33-3p can bind to ADAM10 3' UTR fragment,this suggestted that ADAM10 may be a target gene of miR-33-3p.In addition,the construction of luciferase double reporter gene and RT-PCR as well as western blot methods proved that ADAM10 is the target gene of miR-33-3p.(2)Low selenium diet can upregulate the expression of miR-33-3p and down regulate the ADAM10 expression in chicken kidney tissue,miR-33-3p increased 9 times,and the expression of ADAM10 was down regulated 46%,and the expression of E-cadherin up-regulation,?-catenin,cyclinD1,TCF,c-myc,survivin were down-regulated expression,and the expression of apoptosis gene Bak,Bax and Caspase3 up-regulation,and antiapoptosis gene Bcl-2 was down-regulated expression,and there was a significant difference compared with the control group(P < 0.05),and the kidney apoptosis was significantly increased by ultrastructural observation.The above results showed that the apoptosis induced by selenium deficiency was related to the expressions of miR-33-3p and its target gene ADAM10 in kidney tissue.(3)In order to confirm the effect of miR-33-3p on the cell cycle and apoptosis of chicken embryo primary kidney cells,we established a model of miR-33-3p overexpression and knockdown model in primary chicken kidney cells in vitro.The results showed that the overexpression of miR-33-3p in chicken embryonic primary kidney cells,the cell cycle and apoptosis-related genes expression levels were changed,the expression of cell cycle-related genes(?-catenin,cyclinD1,TCF,c-myc,survivin)were down-regulated.The expression levels of apoptosis-related genes(Bax,Bax and Caspase3)were up-regulated,while Bcl-2 was downregulated.At the same time,the cell cycle and apoptosis were detected by flow cytometry after transfection of miR-33-3p mimic and inhibitor.The results showed that cell cycle arrest increased significantly at G0/G1 phase,and apoptosis rate also increased after transfection of miR-33-3p mimic.However,after down-regulated miR-33-3p expression level,the expression of cell cyclerelated genes(?-catenin,cyclinD1,TCF,c-myc,survivin)were up-regulated.The expression levels of apoptosis-related genes(Bax,Bax and Caspase3)were down-regulated,while Bcl-2 was up-regulated.And the flow cytometry also showed the progress of cells was smooth and the rate of apoptosis was significantly decreased after transfection of miR-33-3p inhibitor.Finally,we also demonstrated by AO-EB method that overexpression of miR-33-3p in chicken embryo primary kidney cells increased the apoptosis rate,while inhibiting the miR-33-3p level in the cells decreased the apoptosis rate.These results suggested that miR-33-3p can induce cell cycle arrest and apoptosis by inhibiting ADAM10 expression.In conclusion,low selenium diet can induce the up-regulation of miR-33-3p expression,and through negative regulation of ADAM10,and further induced cell cycle arrest and apoptosis.This study laid a foundation for further study of the mechanism of action between miR-33-3p and selenium deficiency,and provided a theoretical basis for the prevention and treatment of seleniumdeficient kidney function injury mechanism,and also provided a reference for comparative medicine.