MicroRNA-1 regulates smooth muscle cell differentiation by repressing Kruppel-like factor 4

Abstract

The role of microRNA-1 (miR-1) has been studied in cardiac and skeletal muscle differentiation. However, it remains unexplored in vascular smooth muscle cells (SMCs) differentiation. The aim of this study was to uncover novel targets of and shed light on the function of miR-1 in the context of embryonic stem cell (ESC) differentiation of SMCs in vitro. miR-1 expression is steadily increased during differentiation of mouse ESC to SMCs. Loss-of-function approaches using miR-1 inhibitors uncovered that miR-1 is required for SMC lineage differentiation in ESC-derived SMC cultures, as evidenced by downregulation of SMC-specific markers and decrease of derived SMC population. In addition, bioinformatics analysis unveiled a miR-1 binding site on the Kruppel-like factor 4 (KLF4) 3' untranslated region (3'UTR), in a region that is highly conserved across species. Consistently, miR-1 mimic reduced KLF4 3'UTR luciferase activity, which can be rescued by mutating the miR-1 binding site on the KLF4 3'UTR in the reporter construct. Additionally, repression of the miR-1 expression by miR-1 inhibitor can reverse KLF4 downregulation during ESC-SMC differentiation, which subsequently inhibits SMC differentiation. We conclude that miR-1 plays a critical role in the determination of SMC fate during retinoid acid-induced ESC/SMC differentiation, which may indicate that miR-1 has a role to promote SMC differentiation.

FIG. 1.

miR-1 is upregulated during SMC differentiation. (A) Representative Western blot showed that the pluripotent gene octamer-binding protein 4 (OCT4) was downregulated and SMC-specific markers upregulated dramatically along with the RA treatment on mouse ESCs at the indicated days. (B) miR-1 expression during RA-induced SMCs from ESCs cultured for 0, 3, 6, or 9 days, at which time miR-1 were measured by TaqMan miR assay and normalized to U6. Fold changes are shown with respect to DMSO-treated cells, where miR-1 levels on day 0 were set to a value of 1. miR-1, microRNA-1; SMC, smooth muscle cell; RA, retinoic acid; SMMHC, smooth muscle myosin heavy chain; ESC, embryonic stem cell; α-SMA, α-smooth muscle actin; DMSO, dimethyl sulfoxide.

FIG. 2.

Inhibition of miR-1 represses SMC differentiation. (A, B) Regulation of SMC differentiation by miR-1 inhibitor. About 50 nM miR-1 inhibitor repressed expression of SMC-specific markers, including α-SMA and SMMHC, as evidenced by Western-blotting (C). ^*P < 0.05. (C) miR-1 inhibitor reduced the SMC differentiation efficiency determined by fluorescence-activated cell sorting. Left panel: Green histogram represents IgG2a isotype control and red represents anti-α-SMA of rat aorta SMCs (RASMCs) as positive control. Right panel: the proportion of the α-SMA-positive cells with green and blue representing inhibitor NC and miR-1 inhibitor, respectively. n = 4. PCR, polymerase chain reaction; NC, negative control; FITC, fluorescein isothiocyanate.

FIG. 3.

KLF4 attenuates ESC/SMC differentiation. (A) Representative Western blots probing for KLF4 and MyoCD in ESCs differentiating to SMCs at the indicated days. (B) Mouse ESCs were infected with adenovirus expressing KLF4 (Ad-KLF4) and subsequently induced to differentiate into SMCs with RA. The derived cells were subject to qRT-PCR analysis and the expression of SMC markers was detected in extracts from cells differentiated for 6 days. 18sRNA served as internal control for qRT-PCR. KLF4, Kruppel-like factor 4; MyoCD, myocardin. *P < 0.05.

FIG. 4.

KLF4 is a target of miR-1 during ESC/SMC differentiation. (A) Predicted target site of miR-1 in the 3′UTR of mouse KLF4, with the seed region, and alignment of the homologous regions of the 3′UTR of KLF4 from human, mouse, rat, and dog. Predicted miR-1 binding sites are highlighted in gray. (B) WT and MU reporter constructs were co-transfected into HEK 293 cells with miR mimic or mimic NC as indicated. Individual luciferase activity was normalized to the responding thymidine kinase (TK) promoter–Renilla-luciferase activity. Relative luciferase activities were expressed as mean ± standard deviation. Data shown are representative samples from at least 3 independent experiments, each done in triplicates. ^*P < 0.05. (C) Mouse ESCs were transfected with miR-1 inhibitor or inhibitor NC. KLF4 and MyoCD protein was detected in extracts from cells differentiated for 72 h. β-tubulin served as internal control. 3′UTR, 3′ untranslated region; WT, wild type; MU, mutant.