MicroRNA-196 Regulates HOX Gene Expression in Human Gluteal Adipose Tissue

Abstract

Objective: Lower body fat is associated with diminishing cardiometabolic risk. Physiological differences between gluteofemoral and abdominal subcutaneous adipocyte functions are known, but the molecular basis for depot differences in adipocyte function is poorly understood. The objective of this study was to identify depot differences in microRNA (miRNA) expression in human abdominal and gluteofemoral subcutaneous adipose tissues and their implication in gene regulation.

Methods: Abdominal and gluteofemoral adipose tissue aspirates obtained from 18 participants (9 male and 9 female, age 30 ± 1.5 y, BMI 27.3 ± 1.23 kg/m2 ) were analyzed for miRNA expression profiles by next-generation DNA sequencing. The raw reads were mapped to miRBase 17, and differentially expressed miRNAs were confirmed by qRT-PCR. The hsa-mimic-miR196a was transfected into cultured abdominal preadipocytes isolated from five women with obesity. Target gene expression was evaluated by RT-qPCR.

Results: Among the 640 miRNAs detected in adipose tissue, miR196a2, miR196a1, miR196b, and miR204 showed a higher expression in the gluteofemoral depot (fold change = 2.7, 2.3, 1.7, and 2.3, respectively) independent of sex. Bioinformatic analyses and human primary preadipocyte transfection with miR196 suggested that the differentially expressed miRNAs could directly or indirectly modulate homeobox (HOX) gene expression.

Conclusions: The miR196 gene family could play an important role in the regulation of HOX gene expression in subcutaneous adipose tissue and in fat distribution variation.

Trial registration: ClinicalTrials.gov NCT00704197 NCT01745471.

Conflict of interest statement

Disclosure: The authors have no conflicts of interest and nothing to disclose

© 2017 The Obesity Society.

Figures

Figure 1. MicroRNAs differentially expressed between Abdominal and Gluteal subcutaneous adipose tissue in both sexes (FDR p

Fold change is the ratio between abdominal and gluteal expression. Black bars indicate microRNA selected for qRT-PCR validation. Fold changes were calculated by dividing the average of the means of miRNA copies number in abdominal depot by the average of the means of miRNA copies number in gluteofemoral depot.

Figure 2. miRSequencing quantification for three selected microRNAs differentially expressed between Abdominal (white bars) and Gluteal (grey bars) subcutaneous adipose tissue split by sex

Data are presented as mean ± SEM of females (n=9) and males (n=9). GF: Gluteofemoral depot – Abd: Abdominal depot

Figure 3. Expression of selected microRNAs in sc adipose tissue (qRT-PCR) in male and female combined

A: Confirmation of the miRNA sequencing data by qPCR for miR196a, miR196b and miR204. B: Expression of selected microRNAs trend to be depot specific only in adipocytes. Data are presented as mean ± SEM of n=16 paired abdominal and gluteal tissue samples, n between 2 and 4 for isolated adipocytes and stromal-vascular fractions. * P<0.05 compared with abdominal depot, paired t-test. C: Spearman correlations between miRNA expression in specific depot and clinical data for n=9 women (pink) and n=9 men (black).

Figure 4. Relationships between selected microRNAs and HOX gene expression

The relationships of the differentially expressed HOX genes, miR196a, miR196band miR204 were determined using SEM (see methods for details) to build a co-expression network HOX genes are represented by numbered boxes (not to scale). Genes upregulated in gluteal depot as compared to abdominal are in green; genes down regulated in gluteal depot are in red. Arrows indicate a positive correlation, dashed arrows indicate negative correlation. A: miR196a2 and miR196b showed interactions with HOX A cluster on chromosome 7. B: miR196a1/a2 and miR204 showed interactions with HOX B cluster on chromosome 17, HOX C cluster on chromosome 12 and HOX D cluster on the chromosome 2. C: Expression of HOXA7, HOXB7, HOXB8 and HOXC8 in miR196a transfected abdominal preadipocytes. Bar graph represents mean and SEM of relative expression of miR-196a at 72hrs post transfection. HOX genes relative expression was plotted for each participant, circle represent cells transfected by hsa-Control, square represent cells transfected by hsa-miR196a. Gene expression was normalized to Cyclophilin A. * paired t-test p<0.01. NTC: Negative control