doi: 10.1016/j.omtn.2022.08.013.
eCollection 2022 Sep 13.
miR-539-5p regulates Srebf1 transcription in the skeletal muscle of diabetic mice by _targeting DNA methyltransferase 3b
Affiliations
- PMID: 36090753
- PMCID: PMC9439965
- DOI: 10.1016/j.omtn.2022.08.013
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miR-539-5p regulates Srebf1 transcription in the skeletal muscle of diabetic mice by _targeting DNA methyltransferase 3b
Mol Ther Nucleic Acids.
.
Abstract
Aberrant DNA methylation is associated with diabetes, but the precise regulatory events that control the levels and activity of DNA methyltransferases (DNMTs) is not well understood. Here we show that miR-539-5p _targets Dnmt3b and regulates its cellular levels. miR-539-5p and Dnmt3b show inverse patterns of expression in skeletal muscle of diabetic mice. By binding to the 3' UTR of Dnmt3b, miR-539-5p downregulates its levels in C2C12 cells and in human primary skeletal muscle cells. miR-539-5p-Dnmt3b interaction regulates Srebf1 transcription by altering methylation at CpG islands within Srebf1 in C2C12 cells. Dnmt3b inhibition alone was sufficient to upregulate Srebf1 transcription. In vivo antagonism of miR-539-5p in normal mice induced hyperglycemia and hyperinsulinemia and impaired oral glucose tolerance. These mice had elevated Dnmt3b and decreased Srebf1 levels in skeletal muscle. db/db mice injected with miR-539-5p mimics showed improved circulatory glucose and cholesterol levels. Oral glucose tolerance improved together with normalization of Dnmt3b and Srebf1 levels in skeletal muscle. Our results support a critical role of miR-539-5p and Dnmt3b in aberrant skeletal muscle metabolism during diabetes by regulating Srebf1 transcription; modulating the miR-539-5p-Dnmt3b axis might have therapeutic potential for addressing altered skeletal muscle physiology during insulin resistance and type 2 diabetes.
Keywords: DNA methylation; DNMT3b; Srebf1; diabetes; miR-539-5p; skeletal muscle.
© 2022 The Author(s).
Conflict of interest statement
The authors declare no competing interests.
Figures
Expression of DNMTs and miRNAs is altered in skeletal muscle of db/db mice (A and B) Total RNA was isolated from the skeletal muscles of normal (db/+) and diabetic (db/db) mice, and 1 μg RNA was reverse transcribed and subjected to qRT-PCR to assess the transcript levels of Dnmt3a (A) and Dnmt3b (B). 18S rRNA was used as the loading control. (C and D) Skeletal muscle of normal (db/+) and diabetic (db/db) mice was lysed as described under “Materials and methods;” 40-μg lysates were run on SDS-PAGE, and the levels of DNMT3a (C) and DNMT3b (D) were evaluated by western blot analysis. HSC70 was used as a loading control. Densitometric analyses of the expression are shown below. (E) Skeletal muscle of normal (db/+) and diabetic (db/db) mice was lysed as described under “Materials and methods,” and 20-μg lysates were used to measure DNMT activity. (F) DNMT1 protein levels were assessed in skeletal muscle of db/+ and db/db mice by western blot analyses, where 40 μg protein was resolved on SDS-PAGE and probed with a DNMT1 antibody. HSC70 was used as the loading control. Densitometric analyses of the expression are shown below. (G–I) Total RNA was isolated from skeletal muscle of normal (db/+) and diabetic (db/db) mice, and 1 μg RNA was reverse transcribed and subjected to qRT-PCR for expression of miR-539-5p (G), miR-381-3p (H), and miR-31-5p (I). All experiments were performed in at least four animals in each group, and values are reported as means ± SEM. ∗p < 0.05, ∗∗p < 0.01.
miR-539-5p inhibits Dnmt3b levels by binding to its 3′ UTR (A) Differentiated C2C12 cells were transfected with the scramble (Scr) or mimics of miR-539-5p, miR-381-3p, or miR-31-5p (1–50 nM). Upon termination of incubation at 48 h, cells were lysed, and 40 μg protein was subjected to western blot analysis using a DNMT3b antibody. HSC70 was used as a loading control. Densitometric analyses of the blots are shown below. (B) Differentiated C2C12 cells were transfected with the Scr or the miR-539-5p mimic with or without its inhibitor. After 48 h, transcript levels of Dnmt3b were quantified by qRT-PCR. 18S rRNA was taken as the loading control. (C) Cells incubated as in (B) were lysed, and lysates (40 μg) were assessed for DNMT3b protein levels by western blot analysis using a DNMT3b antibody. HSC70 was used as a loading control. Densitometric analysis of the blots is shown below. (D) Depiction of the miR-539-5p binding site in the 3′ UTR of Dnmt3b and the mutations (red) incorporated in the miRNA binding site of the Dnmt3b 3′ UTR (MT) as described under “Materials and methods.” (E) C2C12 cells were transfected with wild-type (WT) or mutated (MT) 3′ UTR luciferase constructs of Dnmt3b together with the Scr or the miR-539-5p mimic with or without its inhibitor. After 48 h of incubation, cells were harvested, and luciferase activity was measured as described under “Materials and methods.”. Renilla luciferase activity was normalized to firefly luciferase activity. (F) C2C12 cells were transfected with biotin-labeled Scr or biotin-labeled miR-539-5p mimic (50 nM), and after 48 h, cells were harvested, lysed, and pulled down using streptavidin-linked Dynabeads. Enrichment of Dnmt3b mRNA in biotin-labeled Scr- or miR-539-5p mimic-transfected cells was quantified by real-time PCR using Dnmt3b-specific primers. (G) Primary human skeletal muscle cells were transfected with the Scr or the miR-539-5p mimic with or without its inhibitor. Upon termination of incubation (48 h), DNMT3b expression was assessed by western blot analysis using a DNMT3b antibody. HSC70 was used as a loading control. Densitometric analysis of the same is given alongside the blot. All experiments were performed in at least 3 sets for each group. (H and I) 1 μg RNA from skeletal muscle of chow diet- and HFD-fed mice (n = 5) was reverse transcribed and subjected to qRT-PCR to evaluate the expression of miR-539-5p (H) and Dnmt3b (I). Sno234 and 18S rRNA, respectively, were used as normalization controls. Values are means ± SEM. ∗p < 0.05, ∗∗p < 0.01.
Srebf1, Irs1, and Prkab2 harbor CpG islands, and their transcript levels are downregulated in skeletal muscle of db/db mice (A) AMPK and insulin signaling pathways are enriched among the downregulated genes in db/db mouse skeletal muscle, with Prkab2, Srebf1, and Irs1 commonly mapped to both pathways. (B) Skeletal muscle RNA (1 μg) from db/+ and db/db mice was reverse transcribed and subjected to qRT-PCR for transcript expression of Prkab2, Srebf1, and Irs1 using gene-specific primers. 18S rRNA was used as the loading control. Data are from at least four animals in each group, and values are means ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001. (C) Presence of CpG islands (enclosed within red boxes) across the Prkab2, Srebf1, and Irs1 genes as identified using the UCSC Genome Browser.
miR-539-5p regulates Srebf1 transcript levels by inhibiting Dnmt3b expression and altering CpG methylation on the Srebf1 gene in C2C12 cells (A) Differentiated C2C12 cells were transfected with the Scr or the miR-539-5p mimic with or without its inhibitor. After 48-h incubation, cells were harvested, and expression of transcript levels of Prkab2, Srebf1, and Irs1 (as described under “Materials and methods”) was evaluated using qRT-PCR. 18S rRNA was used as the loading control. (B) Schematic of CpG islands across the Srebf1 gene in mice. Each CpG site was randomly split into three regions, and primers were designed to amplify these regions within each CpG island using primers sets (P1, P2, and P3) as shown. (C) Differentiated C2C12 cells were transfected as in (A) with Scr or the miRNA mimic, and upon termination of incubation, genomic DNA was isolated, sheared, and immunoprecipitated (2 μg) with a 5-methylcytosine antibody (2 μg) or non-immune rabbit IgG (2 μg) and incubated overnight at 4°C. Methylation-enriched CpG regions within the Srebf1 gene were quantified by real-time PCR using CpG region-specific primers. (D) Genomic DNA isolated from skeletal muscle of normal db/+ and diabetic db/db mice was sheared, and the methylation status within Srebf1 was quantified by real-time PCR as described in (C). Differentiated C2C12 cells were transfected with the Scr or Dnmt3b siRNA (50–100 nM), and after 48 h, cells were harvested, and DNMT3b expression was quantified at the transcript (E) and protein (F) levels. 18S rRNA and HSC70 were used as normalization controls, respectively. (G) Differentiated C2C12 cells transfected with the Scr or Dnmt3b siRNA (50–100 nM) were harvested, and 1 μg RNA was reverse transcribed to evaluate the transcript levels of Srebf1 by qRT-PCR. 18S rRNA was used as the normalization control. Values are means±SEM of at least three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ns, non-significant.
Dnmt3b inhibition rescues the miR-539-5p inhibitor-induced decrease in Srebf1 levels in C2C12 cells (A and B) C2C12 cells were incubated in the presence of a DNMT3b inhibitor (nanaomycin A [NA]) at doses of 1, 5, and 10 μM. Control cells were incubated in the presence of DMSO. After 48 h, DNMT1, DNMT3a, and DNMT3b levels were evaluated by qRT-PCR (A) and western blot (B). 18S rRNA and HSC70, respectively, were used as the loading controls. D, DMSO. (C) Cells incubated as in (A) were assayed for activity of DNMT as described under “Materials and methods.” Data were normalized to the total protein content. (D) Total RNA was isolated from cells incubated as in (A), and the transcript levels of Srebf1 were quantified using specific primers. 18S rRNA was used as the normalization control. (E–G) C2C12 cells were transfected with the Scr or miR-539-5p inhibitor (25–50 nM), and after 48 h, the status of DNMT3b (E and F) and Srebf1 (G) were evaluated by qRT-PCR and western blot analysis. HSC70 and 18S rRNA were taken as the normalizing controls for western blot and qRT-PCR experiments, respectively. (H and I) C2C12 cells were transfected with the Scr or miRNA-539-5p alone (50 nM) or along with Dnmt3b siRNA (100 nM), and after 48 h, Dnmt3b (H) and Srebf1 (I) transcript levels were evaluated by qRT-PCR. 18S rRNA was taken as the endogenous control. (J) C2C12 cells were transfected with the empty vector or the Dnmt3b overexpression vector (1 μg) together with the Scr or miR-539-5p (50 nM); after 48 h, total RNA was isolated, and the transcript levels of Srebf1 were evaluated by qRT-PCR. 18S rRNA was used as the endogenous control. All experiments were performed at least three times for each group, and values are reported as means ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
In vivo antagonism of miR-539-5p induces hyperglycemia and hyperinsulinemia in mice (A) Schematic of the in vivo experimental pipeline. C57BL/6 mice were injected i.v. with Scr or miR-539-5p antagomir at a dose of 5 mg/kg. Three injections were given, one every other day. An OGTT was done on day 6, and on day 8, mice were euthanized. (B) Blood glucose levels were measured randomly the following day after every injection. (C and D) Fasting blood glucose levels at 6 and 12 h of fasting (C) and circulatory insulin levels (D) were measured in Scr- and miR-539-5p antagomir-injected mice. (E) OGTT was performed on Scr- and miR-539-5p antagomir-injected mice after 12 of fasting. (F and G) Serum triglyceride (F) and cholesterol (G) levels were measured in sera of Scr- and miR-539-5p antagomir-injected mice as described under “Materials and methods.” Data are from at least four animals in each group, and values are means ± SEM. ∗p < 0.05 compared with Scr-injected mice.
In vivo inhibition of miR-539-5p induces DNMT3b expression and inhibits Srebf1 expression in skeletal muscle (A) Total RNA (1 μg) isolated from skeletal muscle of Scr- and miR-539-5p antagomir-injected mice was reverse transcribed and assessed for the transcript levels of Dnmt3b by qRT-PCR. 18S rRNA was used as endogenous control. (B) Skeletal muscle tissue from Scr- and miR-539-5p antagomir-injected mice was homogenized in lysis buffer, and 40 μg protein was run for SDS-PAGE and subjected to western blot analysis for detection of DNMT3b using specific antibodies. HSC70 was used as a loading control. (C) 1 μg of RNA from skeletal muscle tissue of Scr- and miR-539-5p antagomir-injected mice was reverse transcribed, and transcript levels of Srebf1 were evaluated by qRT-PCR. 18S rRNA was used as a normalizing control. (D and E) Total RNA (1 μg) from the livers of Scr- and miR-539-5p antagomir-injected mice was reverse transcribed, and transcript levels of Dnmt3b (D) and Srebf1 (E) were evaluated by qRT-PCR. 18S rRNA was used as a normalization control. (F) Mouse hepatic Hepa 1-6 cells were transfected with the Scr or miR-539-5p mimic (50 nM). After 48 h, cells were harvested to evaluate the expression of Dnmt3b and Srebf1 transcripts by qRT-PCR, normalized to 18S rRNA. Data are from at least 4 animals in each group, and data in (F) are from experiments in three independent sets; values are means ± SEM. ∗p < 0.05 compared with Scr-injected mice.
miR-539-5p administration improves hyperglycemia and normalizes miR-539-5p, Dnmt3b, and Srebf1 levels in skeletal muscle of diabetic db/db mice (A) Schematic of the experimental design for db/+ and db/db animals. db/db mice were injected with the Scr to miR-539-5p mimics (i.v. through the tail vein) at a dose of 5 mg/kg body weight. db/+ mice were injected with Scr. Three injections were given, one every other day. An OGTT was done on day 6, and on day 8, mice were euthanized. (B and C) Random blood glucose levels on the following day after every injection (B) and fasting blood glucose levels at 6 and 12 h of fasting (C) were measured in db/+ and db/db mice. (D) OGTT was performed after an oral glucose dose (2 g/kg body weight), and blood glucose levels were measured at time intervals of 30, 60, 90, and 120 min after the glucose feed. (E and F) Serum triglyceride (E) and cholesterol (F) levels were measured in sera of db/+ and db/db mice. (G–I) Total RNA was isolated from skeletal muscle tissue of db/+ mice injected with the Scr or db/db mice injected with the Scr or miR-539-5p mimics, and the transcript levels of miR-539-5p (G), Dnmt3b (H), and Srebf1 (I) were assessed by qRT-PCR. Sno234 or 18S rRNA was taken as the normalization control. Values are means ± SEM of four animals in each group. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared with db/+ mice injected with Scr; #p < 0.05, ##p < 0.01 compared with db/db mice injected with Scr.
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