miR-129-5p Inhibits Adipogenesis through Autophagy and May Be a Potential Biomarker for Obesity

Abstract

Introduction: Obesity has an unclear pathogenesis. MicroRNAs (miRNAs) may function as biologically active molecules for obesity through regulating adipocyte differentiation. This study aimed to identify how miR-129-5p (a specific miRNA) regulates adipogenesis in vitro and explore its possible role in the pathogenesis of obesity in humans.

Materials and methods: The miR-129-5p expression was detected in obese mouse models. The effect of miR-129-5p on adipocyte differentiation was observed, and the adipose markers were analyzed. Bioinformatics and dual-luciferase reporter assay were applied to predict and confirm the target genes of miR-129-5p. The human serum samples were detected and analyzed.

Results: miR-129-5p is highly expressed in adipose tissues of db/db mice. Gain- and loss-of-function studies show that miR-129-5p could significantly inhibit adipocyte differentiation and white adipocyte browning in vitro and decreases the level of specific markers, such as FABP4, UCP1, and PPARγ, in mature white and brown adipocytes. miR-129-5p directly targets ATG7 which is predicted with bioinformatics and confirmed by dual-luciferase reporter assay. Serum miR-129-5p level was evidently elevated in patients with simple obesity (p < 0.01) and correlates with obesity indices, including BMI (r = 0.407, p < 0.029) and fat percentage (r = 0.394, p < 0.038).

Conclusion: miR-129-5p might target on the ATG7-related autophagy signaling network that regulates white and brown adipogenesis. Importantly, the aforementioned results suggest serum miR-129-5p might be a potential biomarker and therapeutic target for obesity.

Figure 1

miR-129-5p level was highly expressed in EWAT of db/db mice. Relative expression level of different miRNAs in EWAT of db/db mice compared with wild-type mice (WT); n = 10 and ^∗∗∗p < 0.001 compared with WT.

Figure 2

White adipocyte differentiation impaired by microRNA mimic-mediated overexpression of miR-129-5p. (a) Expression levels of miR-129-5p. (b) Oil Red O staining of mature adipocytes. The top two images were captured by a camera; the lower two images were acquired with a microscope at 100x amplification. (c) Relative TG content of cells isolated from EWAT. (d) The time course of miR-129-5p expression during normal adipogenic differentiation was detected, and related white adipogenic genes were qualified by RT-qPCR. (e) Protein levels of the target genes were determined by western blot. (f) Densitometry quantification of western blot. The results of Student's t-test are presented as mean ± SEM of a representative of more than three independent experiments (^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001).

Figure 3

miR-129-5p mimics inhibited browning of SVF from abdominally subcutaneous fat tissues in male mice. SVF from abdominally subcutaneous fat tissues was induced to differentiate into brown adipocytes. (a) Oil Red O staining of mature beige adipocytes. (b) Relative TG content of these cells. (c) The time course of miR-129-5p expression during normal beige adipogenic differentiation and C/EBPα, PPARγ2, and UCP1 gene expressions were quantified by RT-qPCR. (d) Marker proteins and genes of brown adipocytes were determined by western blot. (e) Densitometry quantification of western blot. Data were analyzed with Student's t-test and is presented as mean ± SEM of a representative of more than three independent experiments (^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001).

Figure 4

Overexpression of miR-129-5p impaired brown adipocyte differentiation. (a) Oil Red O staining of mature brown adipocytes. (b) Relative TG content of the mature brown adipocytes. (c) The time course of miR-129-5p expression during normal brown adipogenic differentiation and the expression of regulators involved in adipogenesis were analyzed by RT-qPCR. (d) Protein levels of the target genes were determined by western blot. (e) Densitometry quantification of western blot in (d). The SVF from interscapular fat tissues was induced to differentiate toward the brown adipocytes. Data were analyzed with Student's t-test and were presented as mean ± SEM of a representative of more than three independent experiments (^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001).

Figure 5

ATG7 was directly downregulated by miR-129-5p in vitro. (a) Predicted target genes were validated by RT-qPCR in SVF white adipocytes. (b) Predicted target genes were validated by RT-qPCR in beige adipocytes. (c) miR-129-5p regulatory element in the 3′UTR of mouse ATG7 was identified by Targetscan. MRE, miRNA regulatory element; MRE-wt, wild-type MRE; MRE-mut, mutated MRE. (d) The effect of miR-129-5p mimics on the reporter construct containing ATG7-3′UTR in MRE-wt or MRE-mut as determined in HEK 293T cells. Relative luciferase units (RLU) are shown. (e–g) ATG7 and LC3I/II were determined by western blot transfected control and miR-129-5p mimics in mature white, beige, and brown adipocytes from SVF. Error bars represent SEM of more than three independent experiments (^∗∗p < 0.01 and ^∗∗∗p < 0.001).

Figure 6

The association of serum miR-129-5p with obesity in humans. (a) Relative miR-129-5p expression level was verified by RT-qPCR in the serum samples from the normal weight group (n = 15) and patients with simple obesity (n = 16); ^∗∗p < 0.01 compared with normal weight subjects. (b) Circulating level of miR-129-5p was detected to positively correlate with BMI in humans; n = 31, r = 0.407, and p < 0.05. (c) Serum miR-129-5p was found to be associated with fat percentage in participants; n = 31, r = 0.394, and p < 0.05.