Role of lncRNA LIPE-AS1 in adipogenesis

Abstract

Recent studies have identified long non-coding RNAs (lncRNAs) as potential regulators of adipogenesis. In this study, we have characterized a lncRNA, LIPE-AS1, that spans genes CEACAM1 to LIPE in man with conservation of genomic organization and tissue expression between mouse and man. Tissue-specific expression of isoforms of the murine lncRNA were found in liver and adipose tissue, one of which, designated mLas-V3, overlapped the Lipe gene encoding hormone-sensitive lipase in both mouse and man suggesting that it may have a functional role in adipose tissue. Knock down of expression of mLas-V3 using anti-sense oligos (ASOs) led to a significant decrease in the differentiation of the OP9 pre-adipocyte cell line through the down regulation of the major adipogenic transcription factors Pparg and Cebpa. Knock down of mLas-V3 induced apoptosis during the differentiation of OP9 cells as shown by expression of active caspase-3, a change in the localization of LIP/LAP isoforms of C/EBPβ, and expression of the cellular stress induced factors CHOP, p53, PUMA, and NOXA. We conclude that mLas-V3 may play a role in protecting against stress associated with adipogenesis, and its absence leads to apoptosis.

Keywords: Ceacam1; LIPE; adipogenesis; apoptosis; long non-coding RNA.

Figure 1.

Genomic organization of human LIPE-AS1 and murine mLas genes and expression of mLas-V3 in murine adipose and liver. There is conserved organization and expression of LIPE-AS1 and mLas in mouse and man as shown in the UCSC genome Browser (http://genome.ucsc.edu). (a) UCSC Genome Browser tracks of the human genome (GRCh38/hg38) showing the organization of the LIPE-AS1 variants and their anti-sense genes: LIPE, CXCL17, CEACAM1, and CEACAM8. (b) UCSC Genome Browser tracks of the mouse genome (NCBI/mm9) showing the organization of the three 4732471J01RIK variants (mLas-V1, mLas-V2, and mLas-V3) and their anti-sense genes: Lipe, Cxcl17, Ceacam1, and Ceacam2. (c) UCSC Genome Browser tracks of the mouse genome showing the tissue Long RNA-seq Plus Signal data from the Encode/CSHL data set in mouse tissues where LIPE-AS1 is also shown to be highly expressed in humans. Liver data is included although LIPE-AS1 and mLas expression are low in this tissue. (d) qRT-PCR data of mLas-V3 expression, (e) Ceacam1 expression, and (f) Lipe expression in epididymal adipose tissue compared to liver. For all tissues, qRT-PCR, n = 5 mice. Data normalized to beta-actin using 2^−ΔCt method and analysed using the Student’s t-test.

Figure 2.

Expression of mLas-V3 during adipogenesis. OP9 cells were used as a pre-adipocyte cell model. (a) Images of OP9 cells taken over the 5 days of differentiation. (b) qRT-PCR analysis of mLas-V3 over time course normalized to beta-actin (n = 4). (c) qRT-PCR of mLas-V3 expression two days post start of differentiation in which siRNA knock down of indicated genes was performed at day −1. Data was normalized to beta-actin and compared to siNegative control using the 2^−ΔΔCt method, n = 3 independent experiments. (d) Representative Western blot data confirming knock down of protein expression.

Figure 3.

Nuclear localization of mLas-V3. OP9 cells were fractionated at day 5 of differentiation into nuclear and cytoplasmic RNA fractions and analysed by qRT-PCR. Expression of (a) mLas-V3, (b) nuclear lncRNA Neat1, (c) Ppia and (d) Gapdh cytoplasmic markers. Data normalized to Gapdh using the 2^−ΔCt method, n = 3.

Figure 4.

Knock down of mLas-V3 blocks adipogenesis. OP9 cells were transfected with control or targeting ASOs or mock transfected, subjected to differentiation, and at day 5 were fixed and stained with Oil Red O. (a) Representative images were taken at 10x magnification. (b) Oil Red O staining was quantified by measuring absorbance of solution extracted Oil Red O from day 5 differentiated cells. n = 3 wells.

Figure 5.

Expression levels of mLas-V3, Pparg and Cebpa in mLas-V3 knock down cells at day 2 and day 1 of differentiation. OP9 cells were transfected with control or targeting ASOs or mock transfected. RNA was isolated for qRT-PCR analysis after one and two days post induction of differentiation. (a) Expression of mLas-V3 confirmed knock down at day 2 of differentiation. qRT-PCR of (b) Pparg and (c) Cebpa at day 2. qRT-PCR of levels of expression of (d) mLas-V3, (e) Pparg, and (f) Cebpa in cells treated with ASOs or mock transfected and collected one day after induction of differentiation. Data normalized using beta-actin and compared to Negative A control using the 2^−ΔΔCt method. (g) Protein expression of PPARγ, C/EBPα, and β-actin in day 1 differentiated cells using total cell lysate. (h) Protein expression of total Caspase-3, cleaved Caspase-3, and β-actin in day 1 differentiated cells using cytoplasmic cell lysates.

Figure 6.

Knock down of mLas-V3 in differentiating adipocytes and apoptosis. (a) Time course imaging of transfected cells using CellEvent ReadyProbe Caspase-3/7 reporter. The green signal represents active Caspase-3/7. UT, untreated. (b) Active caspase-3/7 was quantified over the time course. Each dot was counted and data was plotted as ±SEM.

Figure 7.

Protein expression of C/EBβ isoforms and CHOP in cells transfected with mLas-V3 ASOs undergoing differentiation. (a) One day and (b) two days post differentiation. Nuclear protein expression of C/EBPβ and Histone H3 in cells transfected with ASOs and collected (c) one day or (d) two days post differentiation. (e) Nuclear expression of CHOP in cells transfected with ASOs and collected one day post differentiation. (f) Nuclear expression of CHOP in cells transfected with ASOs and collected two days post differentiation.

Figure 8.

Knockdown of mLas-V3 by ASO 5 and 6 lead to activation of different stress pathways. (a) Exon structure of mLas-V3 showing regions targeted by ASO 5 and ASO 6. Image generated using SnapGene software. qRT-PCR levels of expression of (b) mLas-V3, (c) Ddit3, (d) Bbc3, and (e) Pmaip1 in cells treated with ASOs or mock transfected and collected 6 hours post induction of differentiation. qRT-PCR levels of expression of (f) Ddit3, (g) Bbc3, and (h) Pmaip1 in cells treated with ASOs or mock transfected and collected one day post induction of differentiation. Data normalized using beta-actin and compared to Negative A control using the 2^−ΔΔCt method. (i)Protein expression of p53, PUMA, and β-actin in day 1 differentiated cells using total cell lysate.