Identification of a novel lncRNA in gluteal adipose tissue and evidence for its positive effect on preadipocyte differentiation

Abstract

Objective: Peripheral lower body fat is associated with lower cardiometabolic risk. Physiological differences in gluteal compared with abdominal subcutaneous (sc) adipocyte functions are known but the molecular basis for depot differences in adipocyte function is poorly understood. Our goal is to identify novel gene regulatory pathways that underlie the heterogeneity of human fat distribution.

Methods: Abdominal and gluteal adipose tissue aspirates obtained from 35 subjects (age = 30 ± 1.6 years; BMI = 27.3 ± 1.3 kg/m(2) ) were analyzed using Illumina microarrays and confirmed by RT-PCR. The HOTAIR gene was stably transfected into primary cultured human abdominal sc preadipocytes using a lentivirus and effects on adipogenic differentiation were analyzed.

Results: A long noncoding RNA, HOTAIR that was expressed in gluteal but not in Abd sc adipose tissue was identified. This difference was retained throughout in vitro differentiation and was maximal at day 4. Ectopic expression of HOTAIR in abdominal preadipocytes produced an increase in differentiation as reflected by a higher percentage of differentiated cells, and increased expression of key adipogenic genes including PPARγ and LPL.

Conclusions: HOTAIR is expressed in gluteal adipose and may regulate key processes in adipocyte differentiation. The role of this lncRNA in determining the metabolic properties of gluteal compared with abdominal adipocytes merits further study.

Keywords: HOTAIR; adipogenic differentiation; gluteal adipose tissue.

Conflict of interest statement

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

Disclosure Summary: The authors have no conflict of interest to disclose.

Copyright © 2014 The Obesity Society.

Figures

Figure 1. Expression of HOTAIR exclusively in human gluteal sc adipose tissue

Microarray analysis identified HOTAIR as a gene expressed only in gluteal depot (A). HOTAIR expression by qPCR in WAT paired samples (n=35 subjects, paired t-test compared with Abd of same sex *p<0.001 **p<0.0001) (B) and in vitro during differentiation of paired abdominal (ABD) and gluteal (GLUT) human primary preadipocytes (n= 4 independent experiments, paired t-test compared with Abd of same time point *p<0.05) (C). F=female; M=male

Figure 2. Effects of HOTAIR overexpression in abdominal sc preadipocytes

Abd preadipocytes were stably transfected with a control or a pLV-CMV-HOTAIR-mKate2-2A-Puro lentivirus where HOTAIR is under the CMV promoter and mKate2 protein, Puromycin resistance marker and red fluorescent protein are under SV40 promoter. Overexpression of HOTAIR was confirmed by fluorescence microscopy at Day 0 (preadipocyte stage) and Day 14 (adipocyte stage) of the differentiation (A), qPCR analysis showed a 2500-fold increase of HOTAIR gene expression throughout differentiation (A). Brightfield pictures taken at the end of the differentiation showed that the number of differentiated preadipocytes increased in transfected cells (HOTAIR) compared to control cells, (representative picture of n=4 experiments) (B). Sequential increase of gene expression of adiponectin (ADPN) and FABP4 was higher in transfected cells compared to control cells (CTRL) (n=2) (C). Western blot analysis at the end of the differentiation confirmed a higher level of Adiponectin and FABP4 proteins in transfected cells (representative western blot of n=2) (C). HOTAIR transfection leads to an increase of expression of late adipocyte marker genes (Leptin, PPARγ, LPL) and HOXD3 and HOXD4 genes (D). qPCR analysis was performed at the end of the differentiation (D14) and compared with non-transfected cells. Lines connect results from 2 independent set of stable control and HOTAIR cell cultures. Ctrl: Control cells OE: Overexpressed cells.