MicroRNA-27a/b-3p and PPARG regulate SCAMP3 through a feed-forward loop during adipogenesis

Abstract

MicroRNAs (miRNA) modulate gene expression through feed-back and forward loops. Previous studies identified miRNAs that regulate transcription factors, including Peroxisome Proliferator Activated Receptor Gamma (PPARG), in adipocytes, but whether they influence adipogenesis via such regulatory loops remain elusive. Here we predicted and validated a novel feed-forward loop regulating adipogenesis and involved miR-27a/b-3p, PPARG and Secretory Carrier Membrane Protein 3 (SCAMP3). In this loop, expression of both PPARG and SCAMP3 was independently suppressed by miR-27a/b-3p overexpression. Knockdown of PPARG downregulated SCAMP3 expression at the late phase of adipogenesis, whereas reduction of SCAMP3 mRNA levels increased PPARG expression at early phase in differentiation. The latter was accompanied with upregulation of adipocyte-enriched genes, including ADIPOQ and FABP4, suggesting an anti-adipogenic role for SCAMP3. PPARG and SCAMP3 exhibited opposite behaviors regarding correlations with clinical phenotypes, including body mass index, body fat mass, adipocyte size, lipolytic and lipogenic capacity, and secretion of pro-inflammatory cytokines. While adipose PPARG expression was associated with more favorable metabolic phenotypes, SCAMP3 expression was linked to increased fat mass and insulin resistance. Together, we identified a feed-forward loop through which miR-27a/b-3p, PPARG and SCAMP3 cooperatively fine tune the regulation of adipogenesis, which potentially may impact whole body metabolism.

Figure 1

A concept figure for the study. (a) A pipeline implemented to elucidate miRNA–PPARG-target regulatory networks during human adipogenesis. T-bars indicate inhibition; arrows indicate stimulation. (b,c) Expression of miR27a/b-3p and ten predicted targets of miR-27b-3p during adipogenesis. TPM values of GPAM and GPD1 were divided by 10-fold to facilitate visualization in the same graph.

Figure 2

Functional validation of miR-27a/b-3p effects on PPARG, SCAMP3 and ABCA1 in human adipocytes. (a) miR-27a-3p and miR-27b-3p were overexpressed in in vitro differentiated in hASCs and their expression was assessed by RT-qPCR. Results are based on three biological/independent experiments. Expression of genes was normalized to the reference gene SNORD68. (b) miR-27a-3p and miR-27b-3p were overexpressed in in vitro differentiated in hASCs and expression of PPARG, SCAMP3 and ABCA1 was assessed by RT-qPCR. Results are based on three biological/independent experiments. Expression of genes was normalized to the reference gene LRP10. (c,d) miR-27a-3p and miR-27b-3p were overexpressed in in vitro differentiated in hASCs, cells were lyzed to collect the total protein and thereafter proteins were analyzed by Western blot. Results are based on four biological/independent experiments. Expression of SCAMP3 was normalized to the total protein amount. (e) Mimics of miR-27a-3p and miR-27b-3p were transfected together with 3′UTR reporter constructs for SCAMP3, ABCA1 or empty reporter vector in 3T3-L1 cells and changes of luciferase activity was measured in cell lysates. Results are based on two or three biological/independent experiments. Results were analyzed using t-test and presented in fold change ± SD relative to negative control (Neg C). ***P < 0.005, *P < 0.05.

Figure 3

Impact of SCAMP3 on human adipogenesis. (a) Expression of PPARG was knocked down using siRNA in hASCs 24 h before induction of differentiation and until days 2, 6 and 9 of differentiation, upon which the expression of PPARG and SCAMP3 was monitored. (b) Expression of SCAMP3 was knocked down using siRNA in hASCs 24 h before induction of differentiation until days 2, 6 and 9 of differentiation, upon which the expression of SCAMP3 and PPARG was monitored. (c) Expression of SCAMP3 and PPARG was knocked down using siRNA in hASCs 24 h before induction of differentiation until day 9 of differentiation when accumulation of neutral lipids and number of cells was evaluated. (d) Expression of SCAMP3 was knocked down using siRNA in hASCs 24 h before induction of differentiation until days 2, 6 and 9 of differentiation, upon which the expression of FABP4, PLIN1 and ADIPOQ was monitored. Results in A-D are based on four biological/independent experiments. Expression of genes was normalized to the reference gene 18 s. Results were analyzed using t-test and presented in fold change ± SD relative to negative control of a corresponding time point (Neg C). ***P < 0.005, **P < 0.01, *P < 0.05.