miR-1275 inhibits adipogenesis via ELK1 and its expression decreases in obese subjects

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   Figure 1

   miR-1275 is differentially expressed during human adipogenesis and reduced in the visceral adipose tissue of overweight and obese subjects. (A) miR-1275 levels were measured in human visceral preadipocytes (vHPAs) between day 0 and 15 of differentiation (n=3). (B) Expression of miR-1275 over a 15-day period of adipocyte differentiation (n=3); miR-1275 levels were normalized to U6 RNA and then expressed relative to the day-15 value. (C and E) miR-1275 expression, as determined by qRT-PCR, was downregulated in visceral (C) or subcutaneous (E) adipose tissue from overweight and obese human subjects versus lean subjects. (D and F) Linear regression analysis indicated a negative relationship between visceral (D) or subcutaneous (F) adipose tissue miR-1275 levels and body mass index (BMI). Data are expressed as the mean±s.d. *P<0.05, ***P<0.001.
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   Figure 2

   Overexpression of miR-1275 inhibits human preadipocyte differentiation. (A) Representative images of cells stained with oil red O on day 15 of differentiation showing the effect of miR-1275 overexpression. (B) Triglyceride accumulation was quantified at different time points of adipogenesis (n=3). (C, D and E) The relative mRNA levels of adipocyte-specific genes were assessed by qRT-PCR at days 0, 4, 7, 10, and 15. The data represent the mean ± s.e.m. of three independent experiments. *P<0.05, **P<0.01, ***P<0.001. PPARG, peroxisome proliferator-activated receptor gamma; C/EBPA, CCAAT/enhancer-binding protein alpha; SREBP1c, sterol regulatory element-binding protein 1c.
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   Figure 3

   Downregulation of miR-1275 promotes human preadipocyte differentiation. (A) Cellular lipid accumulation was detected by oil-red-O staining at day 15. (B) Triglyceride levels were analyzed at days 4, 7, 10, and 15 (n = 3). (C, D and E) mRNA expression of adipocyte-specific genes was estimated at days 4, 7, 10, and 15. The data represent the mean ± s.e.m. of three independent experiments. *P<0.05, **P<0.01, ***P<0.001.
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   Figure 4

   miR-1275 negatively regulates ELK1 in human visceral adipocytes (vHPAs). ELK1 mRNA levels in preadipocytes transduced with miR-1275 were analyzed by qRT-PCR during adipogenesis at days 0, 4, 7, 10, and 15 (A) and ELK1 protein was measured by Western blot at day 7 (B). (C and D) vHPAs infected with anti-miR-1275 were analyzed by qRT-PCR and Western blot for ELK1 levels at the indicated time points. (E) miR-1275 binding sequences of the wild-type (W.T. ELK1 3′-UTR) and mutant (MUT. ELK1 3′-UTR) reporter vectors. The “seed” match region of miR-1275 is highlighted in bold and the mutant sites are underlined. (F) HEK293T cells were transiently transfected with an miR-1275 mimic or a negative control mimic (CM) combined with a psi-CHECK2 control plasmid (a modified psi-CHECK2 plasmid containing the wild-type ELK1 3′-UTR), or an ELK1 3′-UTR with a mutated binding sequence. Luciferase activity was analyzed 48 h later; for each sample, renilla luciferase activity was normalized to firefly luciferase (n=6). (G) The expression of ELK1 was upregulated in visceral adipose tissue of overweight and obese human subjects. Data are presented as the mean ± s.e.m. of three independent experiments. *P<0.05, **P<0.01.
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   Figure 5

   miR-1275 regulates the expression of KROX20 and KLF5. KROX20 and KLF5 mRNA levels were analyzed by qRT-PCR during adipogenesis at days 0, 4, 7, 10, and 15 in preadipocytes overexpressing miR-1275 (A and B) and in miR-1275 knockdowns (C and D). (E and F) KROX20 and KLF5 expressions were upregulated in visceral adipose tissue from overweight and obese human subjects. Data are presented as the mean ± s.e.m. of three independent experiments. *P<0.05, **P<0.01, ***P<0.001. KROX20, early growth response 2; KLF5, Krüppel-like factor 5.

• © 2016 Society for Endocrinology