Yonsei Med J.  2019 Dec;60(12):1187-1194. 10.3349/ymj.2019.60.12.1187.

MicroRNA-138 Suppresses Adipogenic Differentiation in Human Adipose Tissue-Derived Mesenchymal Stem Cells by Targeting Lipoprotein Lipase

• Wang Y
• Lin L
• Huang Y
• Sun J
• Wang X
• Wang P

• KMID: 2463798
• DOI: http://doi.org/10.3349/ymj.2019.60.12.1187

Abstract

PURPOSE
Adipogenic differentiation of adipose tissue-derived mesenchymal stem cells (AMSCs) is critical to many disease-related disorders, such as obesity and diabetes. Studies have demonstrated that miRNA-138 (miR-138) is closely involved in adipogenesis. However, the mechanisms affected by miR-138 remain unclear. This work aimed to investigate interactions between miR-138 and lipoprotein lipase (LPL), a key lipogenic enzyme, in AMSCs.
MATERIALS AND METHODS
Human AMSCs (hAMSCs) isolated from human abdomen tissue were subjected to adipogenic differentiation medium. Quantitative real-time polymerase chain reaction and Western blot assay were applied to measure the expressions of miR-138, LPL, and the two adipogenic transcription factors cytidine-cytidine-adenosine-adenosine-thymidine enhancer binding protein alpha (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ). The relationship between miR-138 and LPL was predicted utilizing the miRTarBase database and validated by dual luciferase reporter assay.
RESULTS
Showing increases in C/EBPα and PPARγ expression levels, hAMSCs were induced into adipogenic differentiation. During adipogenesis of hAMSCs, miR-138 expression was significantly downregulated. Overexpression of miR-138 by transfection inhibited hAMSCs adipogenic differentiation in vitro. Mechanically, LPL was a target of miR-138. LPL expression was upregulated during adipogenesis of hAMSCs, and this upregulation was reversed by miR-138 overexpression. Functionally, silencing of LPL by transfection exerted similar inhibition of the expressions of C/EBPα and PPARγ. Meanwhile, LPL ectopic expression was able to partly abolish the suppressive effect of miR-138 overexpression on adipogenic differentiation of hAMSCs.
CONCLUSION
Upregulation of miR-138 inhibits adipogenic differentiation of hAMSCs by directly downregulating LPL.

Keyword

miR-138; adipogenic differentiation; hAMSCs; lipoprotein lipase (LPL)

MeSH Terms

Abdomen
Adipogenesis
Blotting, Western
Carrier Proteins
Ectopic Gene Expression
Humans*
In Vitro Techniques
Lipoprotein Lipase*
Lipoproteins*
Luciferases
Mesenchymal Stromal Cells*
Obesity
PPAR gamma
Real-Time Polymerase Chain Reaction
Transcription Factors
Transfection
Up-Regulation
Carrier Proteins
Lipoprotein Lipase
Lipoproteins
Luciferases
PPAR gamma
Transcription Factors

Figure

• 

  Fig. 1 Induction and identification of adipogenic differentiation in human adipose tissue-derived mesenchymal stem cells (hAMSCs). hAMSCs were cultured in adipogenic differentiation medium to induce adipogenic differentiation for the indicated days. (A) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of enhancer binding protein alpha (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ) was conducted. (B) Western blot was used to measure the expression of C/EBPα and PPARγ, and the quantitation was determined with an image analyzer. (C) The expression of miR-138 was assessed by qRT-PCR. Data represent means±standard error of means. *n=3, p<0.05 compared with control hAMSCs on day 0.

• 

  Fig. 2 Overexpression of miR-138 inhibits adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hAMSCs). Overexpression of miR-138 in hAMSCs was obtained by transfection of miR-138 mimic (miR-138) for 2 d. (A) Transfection efficiency was determined with quantitative real-time polymerase chain reaction. (B and C) Expression of enhancer binding protein alpha (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ) mRNA was monitored after adipogenic differentiation induction. (D–F) Expression of C/EBPα and PPARγ protein was monitored after adipogenic differentiation induction via Western blot assay. Data represent means±standard error of means. *n=3, p<0.05 compared with the miR-NC group at the indicated times.

• 

  Fig. 3 miRNA-138 (miR-138) targets lipoprotein lipase (LPL) and negatively regulates LPL expression in human adipose tissue-derived mesenchymal stem cells (hAMSCs). (A) The binding sites between miR-138 and the 3′-UTR of LPL predicted by the miRTarBase database and the mutant are shown. (B and C) Dual-luciferase reporter assay was employed to detect the luciferase activity of wild type of LPL 3′UTR (LPL WT 3′UTR) and mutant of LPL 3′UTR (LPL MUT 3′UTR) after co-transfection with miR-138 or miR-NC. (D and E) Western blot was conducted to measure the effect of miR-138 on LPL protein expression during adipogenic differentiation of hAMSCs. Data represent means±standard error of means. *n=3, p<0.05 compared with the miR-NC group at the indicated times.

• 

  Fig. 4 Silencing of lipoprotein lipase (LPL) suppresses adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hAMSCs). Silencing of LPL in hAMSCs was obtained by transfection with si-LPL for 2 d. (A and B) Expression of LPL was determined through quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot assay. (C and D) The mRNA expression of enhancer binding protein alpha (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ) was examined after adipogenic differentiation induction via qRT-PCR. (E–G) Expression of C/EBPα and PPARγ protein was analyzed after adipogenic differentiation induction utilizing Western blot assay. Data represent means±standard error of means. *n=3, p<0.05 compared with the si-LPL group at the indicated times.

• 

  Fig. 5 Overexpression of lipoprotein lipase (LPL) partly reverses the inhibitory effect of miRNA-138 (miR-138) on adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hAMSCs). Four transfection groups were used to study adipogenic differentiation: miR-NC mimic, miR-138 mimic, miR-138 mimic+pcDNA, and miR-138 mimic+pcDNA-LPL. (A and B) Expression of enhancer binding protein alpha (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ) mRNA was evaluated by quantitative real-time polymerase chain reaction. (C–F) Protein expression of C/EBPα and PPARγ was tested with Western blot assay. Data represent means±standard error of means. *n=3, p<0.05 compared with the si-LPL group or miR-138 mimic+pcDNA group at the indicated times.

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