The role and possible mechanism of lncRNA U90926 in modulating 3T3-L1 preadipocyte differentiation

Figure 1. The expression of lncRNA U90926 during 3T3-L1 adipogenesis. (a) Lipid accumulation was observed with oil red O staining under light microscopy. Magnification 200 ×. (b) Global transcription profiling from our transcriptome microarray analysis on day 0, 2, 4 and 13 during 3T3-L1 preadipocytes differentiation. (c) lnc-U90926 expression was calculated with Bayesian analysis of variance for microarrays (BAMarray; http://www.bamarray.com) by comparing day 2, 4 and 13 versus day 0 undifferentiated preadipocytes. (d) The lnc-U90926 expression was normalized to the level of 18S rRNA with PPARγ2 as a positive control of adipogenesis. Black circles stands for U90926, black squares for PPARγ2. Results are shown as mean±s.e.m. of three independent experiments. *P<0.05, **P<0.01 versus D0.

Figure 2. Lnc-U90926 expression, cellular localization and correlation with obesity. (a) Lnc-U90926 expression in different tissues from C57BL/6 mice compared with WAT. BAT stands for brown adipose tissue, WAT stands for white adipose tissue from epididymal fat, (n=6). (b) Cellular localization analysis: RNA FISH assay of lnc-U90926 in 3T3-L1 preadipocytes. Scale bars, 10 μm. DAPI is 4′, 6-diamidino-2-phenylindole. Lnc-U90926 expression in (c) subcutaneous fat tissue, (d) perirenal fat tissue, (e) epididymal fat tissue extracted from obese mice including high-fat diet induced C57BL/6 obese mice, ob/ob obese mice and db/db obese mice or their parallel control mice. PPARγ2, FABP4 and AdipoQ expression in epididymal fat tissue from (f) HFD obese mice and (g) ob/ob obese mice and db/db obese mice compared with their controls (n⩾3). *P<0.05, **P<0.01, ***P<0.001 versus the control. In panels d–f, HFD stands for high-fat diet induced obese mice; whereas BD stands for mice fed with basal diet, which were used as control for HFD. The control mice for ob/ob obese mice and db/db obese mice were C57BL/6 mice fed with the same high-fat diet with these ob/ob mice and db/db mice.

Figure 3. The effect of lnc-U90926 overexpression on adipogenesis. (a) Representative oil red O staining on day 0, 6, 12 from 3T3-L1 NC and OV cell clones. Magnification, 100 ×. (b) qPCR verification of 3T3-L1 cell lines stably over-expressing lnc-U90926 (designated as OV) and normal control clones (designated as NC). The expression of (c) PPARγ2, (d) FABP4, (e) AdipoQ on day 0 and day 12 during differentiation of 3T3-L1 NC and OV cell clones. (f) The protein expression level of PPARγ and FABP4 in NC and OV cells along with differentiation. The data show the means of three independent experiments, *P<0.05, **P<0.01, ***P<0.001 versus NC.

Figure 4. The effect of lnc-U90926 knockdown on adipogenesis. (a) qPCR verification of knockdown effect of three kinds of lentivirus encoding three different shRNA sequences targeting to lnc-U90926. (b) The expression of PPARγ2, FABP4, C/EBPα and AdipoQ on day 0 and day 6 during the adipogenesis of 3T3-L1 control cells (CON) and knockdown cells (KD) by qPCR. (c, d) Oil red O staining was performed to detect the lipid accumulation in CON and KD cells on day 0 and day 8. (e) The protein expression of PPARγ and FABP4 at different time points during the adipogenesis of CON and KD cells. The results are shown as the mean±s.e.m. of three independent experiments. *P<0.05, **P<0.01, ***P<0.001 versus CON.

Figure 5. The effect of lnc-U90926 overexpression on the transactivation of C/EBPα or PPARγ2. (a) Relative luciferase activity of C/EBPα promoter in HeLa cells. (b) Relative luciferase activity of PPARγ2 promoter. (c) Diagram of PPARγ2 promoter of different lengths. (d) Relative luciferase activity of PPARγ2 promoters with different lengths in HeLa cells. The results are shown as the mean±s.e.m. of three independent experiments. ***P<0.001 versus Vector.