Fig 1: miR-222 inhibits SCD5 expression to regulate the fatty acid desaturation and TG content.a qPCR of endogenous SCD5 mRNA abundance in response to miR222 overexpression and inhibition. n = 3 independent experiments. “***” Indicates statistically significant. b Western blotting of SCD5 protein level in response to miR222 overexpression and inhibition. GAPDH is the internal control. M is the Page Ruler Prestained Protein Ladder 26616 (ThermoFisher Scientific). c Dual luciferase reporter testing of miR222 on SCD5 3'UTR. pRL-TK is the Renilla luciferase expression plasmid used to normalize luciferase activity. n = 3 independent experiments. “***” Indicates statistically significant. d The binding site of miR222 in the SCD5 3'UTR and the introduced mutation. e TG content alteration in response to miR222 overexpression and inhibition in subcutaneous preadipocytes. n = 3 independent experiments. “***” Indicates statistically significant. f Alteration of C16:1/C16:0 and C18:1/C18:0 fatty acid ratio in subcutaneous preadipocytes in response to treatment with miR222 mimics and inhibitor. n = 3 independent experiments. “***” Indicates statistically significant. g TG content in the subcutaneous fat tissue of WT (MSTN+/+) and MSTN-KO (MSTN+/-) pigs (n = 3 biologically independent animals). h TG content in the subcutaneous preadipocytes of WT (MSTN+/+) and MSTN-KO (MSTN+/-) pigs (n = 3 biologically independent animals). i C16:1/C16:0 and C18:1/C18:0 fatty acid ratio in the subcutaneous fat tissue of WT (MSTN+/+) and MSTN-KO (MSTN+/-) pigs (n = 3 biologically independent animals). j C16:1/C16:0 and C18:1/C18:0 fatty acid ratio of subcutaneous preadipocytes of WT (MSTN+/+) and MSTN-KO (MSTN+/-) pigs (n = 3 biologically independent animals). k Representative image of Oil Red O staining of the subcutaneous preadipocytes of WT (MSTN+/+) and MSTN-KO (MSTN+/-) pigs. Scale bar, 100 µm.
Fig 2: MSTN signals through an MEF2C/miR222/SCD5-dependent cascade to affect fatty acid desaturation in pigs.a Expression of MEF2C and SCD5 in response to the restoration of MSTN detected by Western blotting. MSTN restoration is indicated by the presence of flag-tagged MSTN. GAPDH is the internal control. p3×Flag-MSTN is the MSTN expression plasmid that is tagged by the epitope peptide Flag. siNC and siSCD5 show the treatments with negative control siRNA and siRNA against SCD5, respectively. b qPCR detection of miR222 expression in response to the restoration of MSTN. n = 3 independent experiments. “***” Indicates statistically significant. c Ratios of C16:1/C16:0 and C18:1/C18:0 in response to the restoration of MSTN. n = 3 independent experiments. “***” Indicates statistically significant. d A model for the MSTN regulation upon fatty acid metabolism through the MEF2C/miR222/SCD5-dependent cascade.
Fig 3: Identification of miRNA-mRNA regulatory pairs potentially involved in fat deposition.a The schematic of mRNA and miRNA deep sequencing and bioinformatic analysis aimed at identification of potential signaling pathways. b Volcano plot of the differentially expressed mRNAs between MSTN-KO and WT pigs. c Volcano plot of the differentially expressed miRNAs between MSTN-KO and WT pigs. M0 and M1 represent WT and MSTN-KO pigs, respectively. d GO barplot of differentially expressed mRNAs involved in lipid metabolism. e KEGG dotplot of differentially expressed mRNAs involved in lipid metabolism. f qPCR analysis of SCD5 mRNA abundance in response to treatments with five miRNAs. g qPCR analysis of miR222 abundance in the subcutaneous fat tissue of WT and MSTN-KO pigs (n = 3 biologically independent samples, respectively). h Northern blotting of miR222 in WT and MSTN-KO pigs. The yellow arrowhead denotes the position of miR222. Small RNAs were loaded in equal amounts. Probe against miR222 was used as positive control for DIG colorization. M1 and M2 are RiboRuler Low Range RNA Ladder (SM1831, ThermoFisher Scientific) and microRNA Marker (N2102, NEB). i qPCR analysis of endogenous SCD5 abundance in the subcutaneous fat tissue of WT and MSTN-KO pigs (n = 3 biologically independent samples, respectively). j Western blotting of the endogenous SCD5 in the subcutaneous fat tissue of WT and MSTN-KO pigs (n = 3 biologically independent samples, respectively). M is the Page Ruler Prestained Protein Ladder 26616 (ThermoFisher Scientific). k Evolutionary conservation of miR222 in multiple species. Red letters represent the seed sequence of miR222. “*” Represents the evolutionary consensus sequence of miR222. l Binding sites of miR222 on the 3'UTR of SCD5 in five animal species. Red letters represent the annealing region of seed sequence of miR222. “*” Shows the consensus sequence of 3'UTR of SCD5 in the five animal species.
Supplier Page from Abcam for Anti-SCD5 antibody