MiR-205 Mediated Cu-Induced Lipid Accumulation in Yellow Catfish Pelteobagrus fulvidraco

Abstract

The present working hypothesis is that the Cu-induced changes in lipid metabolism may be mediated by miRNAs. Here, we describe the miRNA profile of the liver tissues of yellow catfish exposed to waterborne Cu, based on larger-scale sequencing of small RNA libraries. We identified a total of 172 distinct miRNAs. Among these miRNAs, compared to the control, mRNA expression levels of 16 miRNAs (miR-203a, 205, 1788-3p, 375, 31, 196a, 203b-3p, 2187-5p, 196d, 459-3p, 153a and miR-725, and two novel-miRNAs: chr4-1432, chr-7684) were down-regulated, and mRNA levels of miR-212 and chr20-5274 were up-regulated in Cu-exposed group. The functions of their target genes mainly involved ether lipid metabolism, glycerophospholipid metabolism, linoleic acid metabolism and α-linolenic acid metabolism. Cu exposure inhibited the expression of miR-205, whose predicted target genes were enriched in the pathway of lipid metabolism, including fas, lxrα, ddit3, lamp2, casp3a and baxa. These potential target genes were further verified by Dual-luciferase reporter gene assay. Using primary hepatocytes of yellow catfish, Cu incubation down-regulated miR-205 expression, and increased TG contents and FAS activity. LXR antagonist effectively ameliorate the Cu-induced change of TG content and FAS activity. These data suggest that down-regulation of the miRNA-205 may be an important step in Cu-induced changes in lipid metabolism in yellow catfish.

Keywords: Cu; Pelteobagrus fulvidraco; lipid accumulation; lipid metabolism; miR-205.

Figure 1

miRNA expression between the control and Cu-exposed group. (A) Scatter plot of hepatic miRNA expression levels in control group and Cu-exposed group. Each point represents a miRNA. The X and Y axes showed the normalized expression (log₁₀NE) of miRNAs in each liver tissue. (B,C) Venn diagram compared the expression distribution of mature miRNAs and novel miRNAs between two groups. Numbers represents number and percent of co-expressed or differentially expressed miRNAs.

Figure 2

Hierarchical clustering of differentially expressed miRNAs between the two groups. The heat map was drawn with log₁₀NE of each miRNA. Color from dark to light indicate low frequency to high frequency miRNA between the control and Cu-exposed group. Color map was used to distinguish the difference of expression.

Figure 3

The top 15 KEGG pathway analysis of known miRNAs with differential expression. The horizontal coordinates was the rich factor. The bigger the rich factor the higher the enrichment.

Figure 4

Schematic representation of the miR-205 target sequence within the 3′UTR of fas, lxrα, ddit3, casp3a, baxa and lamp2. (A–F) The binding sites of miR-205 with fas, lxrα, ddit3, casp3a, baxa, and lamp2 3′UTR. “|” indicates nucleotides that are reversely complementary to miR-205. Six nucleotides (complementary to nucleotides 2–7 of miR-205) were mutated to “GACCTA” in the 3′UTR of fas, lxrα, ddit3, casp3a, baxa and lamp2. The numbers indicate the positions of the nucleotides in the reference wild-type sequences.

Figure 5

Fold change of miR-205 after miR-205 mimics transfection in HEK-293T at 24 h. miR-205 fold change levels were measured by Q-PCR. miR-205 expression values were normalized to housekeeping genes (U6) expressed as a ratio of the control. Mean of the results from the control was set as 1. Data represent means ± SEM (n = 3). ** p < 0.01, compared to control, independent-samples T-test.

Figure 6

Activities of the luciferase reporter gene linked to the 3′UTR of fas (A), lxrα (B), casp3a (C), ddit3 (D), baxa (E) and lamp2 (F). The psiCHECK2 luciferase reporter plasmids with the wild-type or mutated 3′UTR sequences of fas, lxrα, ddit3, casp3a, baxa and lamp2 were transiently transfected into HEK-29T cells along with 20 μM miR-205 mimics or negative control. Luciferase activities were measured after 24 h, and firefly luciferase was used for normalization. Mean of the results from the cells transfected by psiCHECK2 vector was set as 1. The data are the mean and standard deviation (SD) of separate transfections (n = 3). * p < 0.05; NS; Student’s t test. The experiment was repeated at least three times.

Figure 7

Cu-induced changes in hepatic lipid droplets of yellow catfish. (A) Images of hepatocytes stained by BODIPY output by fluorescence scanning confocal microscopy. (B) Quantitative analysis of the ratio of fluorescence intensity over area by Image J. Mean of the results from the control was set as 1. The data are mean and standard deviation (SD) of separate transfections (n = 3). * p < 0.05, compared with control. ^# p < 0.05, compared with single LXR antagonists incubation (independent-samples T-test).

Figure 8

Effects of different treatment for 24 and 48 h on miR-205 expression, LXRα protein expression and FAS activity, and TG content in hepatocytes from yellow catfish. (A) Cu exposure inhibited miR-205 expression in vitro at 24 and 48 h. Mean of the results from the control was set as 1. Values are mean ± SEM (n = 3). miR-205 expression values were normalized to U6 expressed as a ratio of the control at 24 and 48h. * p < 0.05; ** p < 0.01; NS: Student’s t test. The experiment was repeated at least three times. (B) Cu incubation increased LXRα protein expression. LXRα protein expression level was increased with Cu incubation in vitro at 24 h, based on the image J analysis. Mean of the results from the control was set as 1. Values are mean ± SEM (n = 4). LXRα protein expression values were normalized to GAPDH protein expressed as a ratio of the control at 24h. (C,D) Change of FAS activity and TG content after miR-205 was inhibited by Cu and lxrα inhibited by LXR antagonist at 24 and 48h. Mean ± SEM (n = 3 independent biological experiments). * p < 0.05, compared with control. ^# p < 0.05, compared with single LXR antagonist incubation (independent-samples t-test).