miR-431 inhibits adipogenic differentiation of human bone marrow-derived mesenchymal stem cells via targeting insulin receptor substance 2

Abstract

Background: An understanding of the mechanism underlying adipogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) will provide new therapeutic approaches for many diseases, including osteoporosis. This study aimed to investigate the role of miR-431 in adipogenic differentiation of hMSCs.

Methods: hMSCs were induced for adipogenic differentiation and miR-431 was detected by polymerase chain reaction (PCR). hMSCs were transfected by miR-431 or small interfering RNA (siRNA) for insulin receptor substance 2 (IRS2). The expression of IRS2 was detected by PCR and Western blot analysis. The targeting of the 3'-untranslated region (UTR) of IRS2 by miR-431 was examined by luciferase assay.

Results: miR-431 expression was decreased during adipogenesis of hMSCs. Overexpression of miR-431 inhibited adipogenic differentiation, accompanied by the downregulation of CCAAT/enhancer binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ), two key regulators of adipogenesis. Moreover, miR-431 decreased both protein and mRNA levels of IRS2. The expression of IRS2 was increased during adipogenic differentiation of hMSCs in conjunction with decreased levels of miR-431, and knockdown of IRS2 in hMSCs inhibited adipogenic differentiation. Luciferase assay confirmed that miR-431 targeted the 3'-UTR of IRS2 in hMSCs.

Conclusions: This is the first study to show that miR-431 inhibits adipogenic differentiation of hMSCs via targeting IRS2.

Keywords: Adipogenic differentiation; Bone marrow-derived mesenchymal stem cells; IRS2; miR-431.

Fig. 1

The expression levels of miR-431 during adipogenesis of hMSCs. Data are presented as mean ± SEM (n = 3). *p < 0.05, **p < 0.01, versus day 0

Fig. 2

miR-431 inhibits adipogenesis of hMSCs. a The expression levels of miR-431 in hMSCs infected by miR-431 lentivirus or negative control (NC) lentivirus. b Oil Red O staining of hMSCs infected by miR-431 lentivirus or NC lentivirus. c The mRNA expression levels of CCAAT/enhancer binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) in hMSCs infected by miR-431 lentivirus or NC lentivirus. d The protein expression levels of C/EBPα and PPARγ in hMSCs infected by miR-431 lentivirus or NC lentivirus. Data are presented as the mean ± SEM (n = 3). **p < 0.01, versus NC

Fig. 3

miR-431 targets the 3′-UTR of IRS2. a The mRNA expression levels of insulin receptor substance 2 (IRS2) in hMSCs infected by miR-431 lentivirus or negative control (NC) lentivirus. b The protein expression levels of IRS2 in hMSCs infected by miR-431 lentivirus or NC lentivirus. c The matching of miR-431 with IRS2 3’-untranslated regions (UTRs). d Luciferase activity assay; HEK293 cells were infected by miR-431 lentivirus or NC lentivirus and transfected with wild-type (WT) or mutant (MUT) IRS2 3′-UTR reporter. Data are presented as the mean ± SEM (n = 3). *p < 0.05, **p < 0.01, versus NC

Fig. 4

The expression levels of IRS2 during adipogenesis of hMSCs. a The mRNA expression levels of insulin receptor substance 2 (IRS2) during adipogenesis of hMSCs. b The protein expression levels of IRS2 during adipogenesis of hMSCs. Data are presented as the mean ± SEM (n = 3). **p < 0.01, versus day 0

Fig. 5

IRS2 promotes adipogenesis of hMSCs. a The mRNA expression levels of insulin receptor substance 2 (IRS2) in hMSCs infected by IRS2 small interfering RNA (siRNA) lentivirus or negative control (NC) lentivirus. b The protein expression levels of IRS2 in hMSCs infected by IRS2 siRNA lentivirus or NC lentivirus. c Oil Red O staining of hMSCs infected by IRS2 siRNA lentivirus or NC lentivirus. d The mRNA expression levels of CCAAT/enhancer binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) in hMSCs infected by IRS2 siRNA lentivirus or NC lentivirus. e The protein expression levels of C/EBPα and PPARγ in hMSCs infected by IRS2 siRNA lentivirus or NC lentivirus. Data are presented as the mean ± SEM (n = 3). *p < 0.05, **p < 0.01, versus NC

Fig. 6

IRS2 rescues the adipogenesis of hMSCs inhibited by miR-431. a The mRNA expression levels of CCAAT/enhancer binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) in hMSCs infected by miR-431 lentivirus alone (miR431) or combined with insulin receptor substance 2 (IRS2) expression vector (miR431 + IRS2). b The protein expression levels of C/EBPα and PPARγ in hMSCs infected by miR-431 lentivirus alone (miR431) or combined with IRS2 expression vector (miR431 + IRS2). Data are presented as the mean ± SEM (n = 3). *p < 0.05, versus miR-431 lentivirus alone