| Excessive accumulation of lipids is a bottleneck problem facing grass carp(Ctenopharyngodon idella)culture in China,which seriously affects the growth performance and quality of grass carp.Selenium is a free radical scavenger of the organism and plays an important role in maintaining the balance of reactive oxygen species(ROS)in the organism,and it is also an important regulator of lipid metabolism in the organism.As an important modality of post-transcriptional regulation,the role of mi RNAs in lipid metabolism has received wide attention.Exosomes(Exo),as intercellular communication tools,play important roles in many physiological processes because they carry mi RNAs,proteins and cytokines.Is the expression of mi RNAs in grass carp liver affected by selenium? Whether exosomes and mi RNAs are involved in the regulation of lipid metabolism by selenium in grass carp is unclear.In this study,we used grass carp as a research object,and explored the mechanism of exosomes and mi RNAs involved in selenium to alleviate high-fat diet induced lipid accumulation in grass carp by using mi RNAs sequencing and cell culture.The main findings were as follows.Experiment 1.Effect of selenium on lipid accumulation in grass carp liver and the expression of mi RNAs.In this study,the transcript levels of co-expressed mi RNAs were compared between the high-fat diet(HFD)and high-fat diet supplemented with selenium(HFD＿Se)groups by bioinformatics analysis.Compared to HFD,two mi RNAs(mi R-22,mi R-100)were found to be significantly up-regulated(P<0.05)and two mi RNAs(mi R-430,mi R-455)were significantly down-regulated(P<0.05)in HFD＿Se.Among them,mi R-22 and mi R-100 were most abundantly expressed.To verify the reliability of mi RNAs sequencing,mi RNAs expression was detected by real-time fluorescence quantitative PCR,and the results were all consistent with the sequencing results,which proved the reliability of this mi RNAs sequencing.In addition,KEGG enrichment analysis revealed that mi R-22 was enriched in energy metabolism-related pathways such as m TOR and insulin signaling pathway.Therefore,it is speculated that mi R-22 may be a key factor involved in selenium regulation of lipid metabolism in grass carp liver.Experiment 2.Validation of mi R-22 and lipid metabolism-related target genes.In this study,based on bioinformatics prediction and combined with the joint screening of grass carp liver transcriptome,the 3’UTR sequence of fibroblast growth factor receptor 1(FGFR1)was found to contain a potential binding site with mi R-22,and wild-type recombinant plasmids and mutant recombinant plasmids were constructed to perform dual fluorophore reporter gene assay.The results showed that mi R-22 significantly inhibited the fluorophore fluorophore activity of the wild-type recombinant plasmid(FGFR1 WT)(P<0.05),but had no significant effect on the fluorophore activity of the mutant recombinant plasmid(FGFR1 MUT)(P>0.05).The mi R-22 NC had no significant effect on the fluorophore activity of both FGFR1 WT and FGFR1 MUT(P>0.05).Therefore,FGFR1 is a direct target gene of mi R-22.Experiment 3.The mi R-22 was involved in the mechanism of selenium alleviation of lipid accumulation in grass carp hepatocytes.In this study,lipid accumulation in grass carp hepatocytes was induced by using oleic acid(OA),and after the successful establishment of a grass carp hepatocyte lipid accumulation model,treatment with sodium selenite was performed,and the changes of triglyceride(TG)content,m RNA levels of genes related to lipid metabolism and other indicators in grass carp hepatocytes were detected.The results showed that 400 μM OA treatment of hepatocytes for 24 h could significantly increase the TG content of grass carp hepatocytes(P<0.05),while 100 μM sodium selenite treatment for 24 h could significantly decrease the TG content of grass carp hepatocytes after OA induction(P<0.05).To investigate the function of mi R-22 in lipid metabolism of grass carp hepatocytes,lipid metabolism-related indexes were detected by transfecting mi R-22 mimic after upregulating mi R-22 levels.The results showed that sodium selenite or mi R-22 mimic treatment could significantly increase mi R-22 levels in grass carp hepatocytes,but the m RNA levels of its target gene FGFR1 were significantly decreased.Moreover,the TG level in grass carp hepatocytes was significantly reduced by sodium selenite or mi R-22 mimic treatment(P<0.05).Further assays showed that sodium selenite or mi R-22 mimic treatment significantly reduced the protein levels of FGFR1,PI3 K,AKT and m TOR and m RNA levels of lipid synthesisrelated genes DGAT1 b,ACC1,PPARγ,FAS and SREBP1 in grass carp hepatocytes(P<0.05).In addition,the effect of sodium selenite in reducing lipid accumulation in grass carp hepatocytes was attenuated after treatment with mi R-22 inhibitor.The above results suggest that mi R-22 may affect the expression of lipid synthesis-related genes and proteins by inhibiting the FGFR1-PI3K-AKT-m TOR signaling pathway,which in turn is involved in the process of selenium alleviation of lipid accumulation in grass carp hepatocytes.Experiment 4.Exosomal mi R-22 mediates the mechanism of selenium reducing lipid accumulation in grass carp.In this study,three hepatocyte-derived exosomes(Exo)were successfully isolated and identified: control hepatocyte exosome(Con-exo),hepatocyte exosome after OA-induced lipid accumulation(OA-exo)and hepatocyte exosome after OAinduced treatment with sodium selenite(Se-exo).The effects of Se-exo treatment on lipid accumulation in grass carp hepatocytes after OA-induced lipid accumulation were investigated.The results showed that the exosomal mi R-22 levels were significantly lower in the OA-exo group than in the Con-exo group(P<0.05),and the exosomal mi R-22 levels were significantly higher in the Se-exo group than in the OA-exo group(P<0.05).PKH-26 dye-labeled exosomes were around the nuclei of hepatocytes,indicating that the exosomes could be taken up by grass carp hepatocytes.Moreover,Se-exo treatment was able to significantly reduce the TG content of OA-induced grass carp hepatocytes(P<0.05).The above results suggest that exosomes may be involved in selenium reduction of OA-induced lipid accumulation in grass carp hepatocytes by carrying mi R-22.In conclusion,selenium targeted the inhibition of FGFR1 through upregulation of mi R-22 levels,which in turn alleviated the high-fat induced hepatic lipid accumulation in grass carp.Moreover,hepatocyte exosomes may be involved in selenium reduction of OA-induced lipid accumulation in grass carp hepatocytes by carrying mi R-22. |
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