| As an important trace element,selenium plays its role mainly in the form of selenocysteine in selenoproteins.Selenoprotein F is one of the 25 selenoproteins in humans.It is an endoplasmic reticulum-resisdent protein and mainly involved in the quality control of the processing and folding of glycoproteins in the endoplasmic reticulum.However,its detailed biological function is still not clear.It is reported that Selenoprotein F knockout mice developed nuclear cataracts but the pathogenic mechanism is still unkown.In this study,Selenoprotein F knockout mice were established with CRISPR/Cas9 gene editing technology at first.Then proteomics analysis of proteins from lens and livers of selenoprotein F knockout mice were performed by iTRAQ(Isobaric Tags for Relative and Absolute Quantification)technology.On the basis of the proteomics analysis,the effects of Selenoprotein F on lens epithelial cell differentiation as well as glucose and lipid metabolism in the liver were studied.The main results are as follows:(1)CRISPR/Cas9 gene editing technology was used on C57/BL6 background to establish Selenoprotein F knockout mice.The mouse had a frameshift mutation caused by several bases deletion in exon 2 and was unable to express normal selenoprotein F.The systemic knockout of selenoprotein F was successfully achieved,which was verified at the gene level and protein level,respectively.The Selenoprotein F knockout mice are viable and fertile,and the phenotype is basically consistent with previous reports.(2)Proteomic analysis of the lens proteins from Selenoprotein F knockout mice was performed with iTRAQ technology.In total,44 up-regulated and 14 down-regulated proteins were screened.Proteomic analysis of differentially expressed proteins was performed in multiple perspectives,including gene ontology analysis,KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analysis,and protein interaction network analysis.It showed that selenoprotein F knockout affected some proteins related to lens development and cell differentiation,such as Filensin and Phakinin,as well as some proteins involved in gap junctions and tight junctions.Some metabolism-related proteins were also affected by Selenoprotein F knockout.(3)Based on the results of lens proteomic analysis,RNA interference was used in human lens epithelial cell lines to achieve Selenoprotein F gene knockdown.The effects of Selenoprotein F knockdown on lens epithelial cell differentiation were studied.The results showed that Selenoprotein F gene knockdown affected the differentiation process of lens epithelial cells induced by b FGF.The up-regulation of some differentiation markers like N-cadherin and ?6-integrin induced by b FGF were inhibited by Selenoprotein F knockdown.Besides,b FGF stimulated signaling pathways were also affected by selenoprotein F knockdown.(4)Proteomic analysis of the hepatic proteins from Selenoprotein F knockout mice was performed with iTRAQ technology.In total,19 up-regulated and 64 down-regulated proteins were screened.Proteomic analysis showed that Selenoprotein F knockout mainly affected some metabolism-related proteins,especially glucose metabolism.And it is also hinted that Selenoprotein F might be related to cancer,diabetes and other diseases.(5)Based on the results of hepatic proteomic analysis,the effects of Selenoprotein F knockout on glucose and lipid metabolism in mice were studied.The results showed that Selenoprotein F knockout mice had glucose intolerance,which may be attribute to insufficient insulin secretion.Moreover,Selenoprotein F knockout mice showed a more significant abnormality of glucose and lipid metabolism under the induction of high fat diets.Selenoprotein F knockout mice exacerbated obesity,hyperglycemia,high blood cholesterol,and hepatic steatosis induced by high fat diets.Proteins levels of Lpl and Ces1 D in the livers of knockout mice were significantly reduced.The deficiency of Selenoprotein F significantly aggravated the disorder of glucose and lipid metabolism induced by high fat diets. |
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