MiR-205 Mediated Cu-induced Lipid Accumulation In Liver Of Yellow Catfish Pelteobagrus Fulvidraco

MicroRNA?miRNA?are 18-nt-long to 25-nt-long noncoding small noncoding RNAs that are conservative and widely expressed in organisms.MiRNA regulate targets gene at the transcriptional and translational levels.MiRNAs play an important role in growth,autophagy and apoptosis.Previous researches suggest that most miRNAs regulate the target genes involved in lipid metabolism by post – transcriptional level in fish.At the translational levels,miRNA represses protein-coding genes via seed pairing to the 3? untranslated regions.Copper?Cu?is a necessary trace element for fish and it participates in various physiological process in fish.However,it can exert adverse toxicological effects when it is present in excess.The previous study showed that Cu has effects on lipid metabolism,but the underlying mechanisms are still not clear.Whether does Cu influence mRNA expression of miRNAs in yellow catfish? Whether do miRNAs mediate the effects of Cu on lipid metabolism? The present study explored the effect of waterborne Cu exposure on miRNA expression and lipid metabolism in the livers of yellow catfish.Then,using primary hepatocytes of yellow catfish,we investigated the mechanism of miR-205 mediating Cu-induced changes of lipid metabolism.The main results of the present study are as follows:1 Effect of waterborne Cu exposure on miRNA expression in yellow catfishIn order to investigate the effects of copper-sensitive miRNA on hepatic lipid metabolism in yellow catfish,they were exposed to two nominal Cu concentrations of zero?control?and 60 ?g/L?Cu-treated group?for 60 days,respectively,with triplicates for each concentration.We constructed two miRNA libraries by using Solexa sequencing.We identified 171 mature miRNAs in the control and 165 mature miRNAs in the Cu-treated group.Among these mature miRNAs,164 miRNAs were co-expressed in both the control and Cu-treated group.In total,16 miRNAs were differentially expressed between the control and Cu-treated group.Compared to the control,2 miRNAs?miR-212 and chr20₅274?were up-regulated and 14 miRNAs?miR-203 a,miR-205,miR-153 a,miR-1788-3p,miR-375,miR-203b-3p,chr21₇684,miR-31,miR-196 a,miR-2187-5p,chr4₁432,miR-196 d,miR-459-3p and miR725?were down-regulated?P< 0.05?,and mRNA levels of miR-212 and chr20-5274 were up-regulated in Cu-exposed group?P<0.05?.We first used an in silico approach to identify the predicted miRNAs targets by using TargetScanFish 6.2,the database of conserved 3?UTR miRNA targets.The result show that 16 differentially expressed miRNAs had 457 targets,and miR-205 had 42 targets.We analyzed the distribution of functional annotation for all targets of differentially expressed miRNAs using GO terms and KEGG.Most of targets were enriched in Biology Process?BP?and the largest number of targets in cell,cell part and binding.Results of KEGG annotation showed that targets of differentially expressed miRNAs were rich in ether lipid metabolism,glycerophospholipid metabolism,linoleic acid metabolism,alpha-linolenic acid metabolism,RNA degradation and amphetamine addiction.Since most of the targets of miR-205 were enriched in the pathway of the lipid metabolism,we speculated that miR-205 mediated Cu-induced changes in the lipid metabolism.Therefore,in the following studies,we place special emphasis on miR-205.2 The determination of target genes involved in the lipid metabolism for miR-205To identify the genes through which miR-205 exerts its effects on lipid metabolism,we first used Targetscan Fish 6.2 to predict target genes related to lipid metabolism and the results were compared with the database of hepatic transcriptome of yellow catfish.The luciferase reporter assay was used to verify that the effect of the miR205 is due to direct interaction with the binding sites of the 3?UTRs.400 – 600 bp wild type DNA fragments of the 3?UTRs of fas,lxr?,ddit3,lamp2,casp3 a and baxa mRNA containing the putative binding sites of miR-205 were synthesized by PCR.The fragments were then subcloned into the XhoI and NotI sites downstream of the Renilla luciferase coding region in the psiCHECK-2 vector and ligated.Mutant recombinant plasmid was only mutates 6mer which was complementary to miR-205 seed sequence to GACCTA.Co-transfection with miR-205 mimics had a marked repressive effect on wild type RL-fas,RL-lxra,RL-casp3 a,RL-ddit3,RL-baxa and RL-lamp2.To further validate the direct interaction between miR-205 and their 3?UTR,we introduced six mutations in the seed sequence of their 3?UTR that is complementary to miR-205.Mutation of the miR-205 sites in fas,lxra,casp3 a,ddit3,baxa and lamp2 prevented the down-regulation of the reporter activities by miR-205,validating that the effect of the miRNA is due to direct interaction with the binding sites of the 3?UTRs.Taken together,these results strongly suggested that miR-205 could inhibit the expression of fas?1031-1052 bp?,lxr??40-61 bp?,ddit3?171-192 bp?,lamp2?621-642 bp?,casp3a?542-563 bp?and baxa?406-427 bp?by binding to the target sites of their 3?UTR.3 The mechanism of miR-205 mediating Cu-induced lipid accumulationHepatocytes were isolated from P.fulvidraco,for the experiment,four groups were designed as follows: Control,LXR antagonist?10 ?M?,Cu?10 ?M?,LXR antagonist?10 ?M?+ Cu?10 ?M?.Compared to the control,Cu incubation down-regulated expression level of miR-205 at both 24 and 48 h?P < 0.05?,but LXR antagonist did not have significant influence on miR-205 expression at 24 and 48 h?P > 0.05?.Cu incubation significantly up-regulated LXR? protein expression?P < 0.05?.Compared to the control,Cu incubation significantly increased TG contents and FAS activities at 24 and 48 h?P < 0.05?.Compared to LXR antagonist,Cu and LXR antagonist coincubation increased TG contents and FAS activities at 24 and 48 h?P < 0.05?.Compared to the control,Cu incubation also significantly up-regulated mRNA levels of fas and ddit3 at 24 and 48 h?P < 0.05?,but did not significantly influence mRNA expression of lamp2,casp3 a and baxa?P > 0.05?.Cu incubation resulted in the increase of the amount and volume of lipid droplets in hepatic cells at 24 and 48 h?P < 0.05?,LXR antagonist reduced lipid droplets in hepatic cells on 24 and 48 h?P < 0.05?.Compared to the LXR antagonist alone,Cu + LXR antagonist co-incubation increased the amount and volume of lipid droplets in hepatic cells at 24 and 48 h?P < 0.05?.Therefore,our data showed that miR-205 down-regulates LXR? expression,suggesting that LXR? is the major target through which miR-205 mediates its effects on Cu-induced changes of lipid metabolism.In conclusion,Cu exposure inhibited the expression level of miR-205 in yellow catfish.The inhibitory effect of miR-205 on translational level of targets gene?lxra,fas?was attenuated after miR-205 was inhibited by Cu,which could lead to the activity of fatty acid synthase and TG content was increased.That causes direct intracellular lipid accumulation.That the inhibitory effect on ddit3,casp3 a,baxa,and lamp2 were attenuated in Cu stress,which could influence of lipid metabolism through endoplasmic reticulum stress,apoptosis,autophagy and other pathways,indirectly affect the lipid metabolism in yellow catfish.We provide evidence that miR-205 mediated the Cu-induced changes of lipid metabolism by targeting lxra.