Repression of versican expression by microRNA-143

Abstract

Smooth muscle cells (SMCs) retain remarkable plasticity to undergo phenotypic modulation in which the expression of smooth muscle markers is markedly attenuated while conversely expression of extracellular matrix (ECM) is dramatically up-regulated. Myocardin is perhaps the most potent transcription factor for stimulating expression of smooth muscle-specific genes; little is known, however, about whether myocardin can orchestrate ECM expression to act in concert with smooth muscle differentiation program. In this study, we demonstrated myocardin coordinate smooth muscle differentiation by inducing transcription of microRNA-143 (miR-143), which attenuates ECM versican protein expression. Previous studies have shown that versican is a chondroitin sulfate proteoglycan of the ECM that is produced by synthetic SMCs and promotes SMC migration and proliferation. Our data demonstrated that myocardin significantly represses versican expression in multiple cell lines, and this occurs through the induction of miR-143 by myocardin. By a modified reverse transcribed PCR, we found that miR-143 specifically binds to the 3'-untranslated region of versican mRNA. Reporter assays validated that miR-143 targets versican 3'-untranslated region through an evolutionarily conserved miR-143 binding site. Furthermore, overexpression of miR-143 significantly represses versican expression, whereas conversely, depletion of endogenous miR-143 results in up-regulation of versican expression. In addition, we demonstrated that myocardin represses versican through induction of miR-143. Finally, we found that the regulation of versican by miR-143 is involved in platelet-derived growth factor BB-induced SMC migration. This study provides the first evidence that myocardin, in addition to activating smooth muscle-specific genes, regulates ECM expression through induction of microRNAs during smooth muscle differentiation.

FIGURE 1.

Effects of overexpression of myocardin on versican gene expression. A, adenovirus encoding GFP or myocardin (myocardin and GFP are encoded on the same adeno-vector by two independent expression cassettes) was transduced into mouse 10T1/2 fibroblast cells for 72 h, and pictures were taken under fluorescence microscope. Note that cell morphology is profoundly altered after overexpression of myocardin. B, adenovirus encoding GFP or myocardin was transduced into mouse 10T1/2 cells, mouse primary aortic vascular SMCs, rat A10 SMCs, and monkey kidney fibroblast COS7 cells, as indicated. After 72 h of infection, cells were harvested with TRIzol for total RNA, and qRT-PCR was conducted to examine endogenous versican expression. *, p < 0.05. C, adenovirus encoding GFP or myocardin was transduced into mouse 10T1/2 cells for 72 h, and then protein lysate was prepared for Western blotting with the indicated antibodies. The equal level of GFP and myocardin transduction is indicated by the expression of GFP immunosignal. Overexpression of myocardin resulted in down-regulation of versican expression at both mRNA and protein levels. Error bars, S.E.

FIGURE 2.

Effects of knocking down myocardin level on versican gene expression. A, A10 cells were infected with adenovirus encoding DN myocardin or a GFP control as indicated. 72 h following infection, total RNA was prepared from infected cells and analyzed by qRT-PCR. Overexpression of DN-myocardin significantly inhibits smooth muscle gene expression, whereas it increases versican expression. B, A10 cells were transfected with siRNA duplex against SRF or scrambled siRNA duplex as a control. 72 h following transfection, total RNA was harvested from cells using TRIzol reagent, and RT-PCR was performed to measure endogenous SM α-actin, SRF, and versican mRNA expression. C, siRNA duplex against myocardin or control siRNA duplex was transfected into A10 SMCs, and total RNA was harvested for qRT-PCR as described in B. Changes in expression that were statistically different from the siRNA control (set to 1) are designated by an asterisk (p < 0.05, Student's non-paired t test). D, silencing of myocardin or SRF in A10 smooth muscle cells was performed as described in B and C. 48 h following silencing, duplex transfection cells were harvested for Western blotting with anti-versican antibody. Western blotting data are representative of three independent experiments. E, densitometry of versican or vinculin was measured, and the relative expression of versican was plotted as (silencing of myocardin or SRF/vinculin)/(silencing of control/vinculin). The relative expression of versican in the silencing control group was set to 1. *, p < 0.05. Knockdown of SRF and myocardin resulted in a significant up-regulation of versican expression in SMCs. Error bars, S.E.

FIGURE 3.

Myocardin induces miR-143 transcription. 10T1/2 cells were transduced with adenovirus encoding myocardin or GFP as a control for 72 h. Subsequently, cells were harvested with TRIzol, and primary (pri) (A), precursor (pre) (B), and mature form of miR-143 (C) were measured by qPCR as indicated. Overexpression of myocardin can significantly induce miR-143 transcription. Error bars, S.E.

FIGURE 4.

miR-143 binds to versican 3′-UTR. A, schematic diagram for the RT-PCR method to determine the interaction of miR-143 with versican 3′-UTR. An oligonucleotide corresponding to miR-X is utilized as a reverse primer to perform the RT reaction, and subsequently the RT product was amplified with a pair of gene-specific primers by PCR. CDS, coding sequence; UTR, untranslated region. B and C, RT-PCR analysis of versican and house-keeping gene RPLP0 using a miR-143 or miR-145 oligonucleotide to prime first-strand synthesis. Following extraction of total RNA from primary rat aortic SMCs, RT was conducted with either a DNA oligonucleotide corresponding to miR-143 or miR-145, a random hexamer (positive control), or no primer (negative control), as indicated. Subsequently, PCR was performed with primers specific to versican, house-keeping gene PRLP0, or water, and the PCR product was separated in agarose gel and visualized with ethidium bromide staining. A 100-bp DNA marker was loaded as a reference. By this modified RT-PCR approach, miR-143 was found to specifically bind to versican 3′-UTR.

FIGURE 5.

miR-143 suppresses versican expression. A, mimic RNA oligonucleotides for scrambled control, miR-143, and miR-133a were transfected into 10T1/2 cells for 72 h. Subsequently, cells were harvested with TRIzol, and qRT-PCR was performed to measure versican and SPP1 mRNA expression (A), or cell protein was collected for Western blot to detect versican protein expression (B), and the relative expression of versican from three independent experiments was quantified and plotted in C. *, p < 0.05. Overexpression of miR-143 results in a significant down-regulation of versican expression at both mRNA and protein levels. D, the scrambled miRNA inhibitor control or miR-143 inhibitor was transfected into A10 SMCs for 2 days. Cells were harvested for qPCR to measure miR-143 expression or qRT-PCR to examine versican mRNA expression. Protein lysates were also collected from the transfected cells for Western blot analysis to measure versican protein expression (E). The relative expression of versican from three independent experiments was quantified and plotted in F. *, p < 0.05. Knockdown of endogenous miR-143 led to a significant up-regulation of versican expression, and this enhancement effect can be seen more dramatically at the protein level. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Error bars, S.E.

FIGURE 6.

miR-143 directly targets versican 3′-UTR. A, schematic diagram of the luciferase reporters containing wild type (WT), deletion, or mutation of the putative miR-143 binding site in the 3′-UTR of versican. The boxed region indicates an evolutionarily conserved “seed” sequence among vertebrate species for miR-143 target recognition. The strategy for generating deletion or mutation of versican 3′-UTR reporter is shown at the bottom. B, 10T1/2 cells were co-transfected with miR-143 or miR-365 expression plasmid and the luciferase reporter constructs harboring the wild type, mutation, or deletion of the predicted miR-143 binding site of versican 3′-UTR, as indicated. Co-transfection of miR-143 significantly inhibits luciferase activity of the reporter harboring wild type versican 3′-UTR, which was abolished when the potential miR-143 target site was mutated or deleted. *, p < 0.05. C, a wild type mouse versican 3′-UTR luciferase construct or a mutant construct containing the mutation of a potential miR-143 binding site and a minimal thymidine kinase-driven Renilla luciferase internal control plasmid were transfected into A10 SMCs, and luciferase activity was measured. The data presented are the luciferase activity normalized with Renilla internal control and mean ± S.E. of six samples. Statistical differences in the activity of the two reporter genes are indicated by an asterisk (p < 0.001). Mutation of the putative miR-143 binding site in the versican 3′-UTR reporter results in increasing luciferase activity. D, A10 cells were transfected with either control miRNA inhibitor (Anti-control), miR-143 inhibitor (Anti-miR-143), or miR-145 inhibitor (Anti-miR-145) for 12 h and then were transfected with luciferase reporter containing wild type or miR-143 binding site mutant of versican 3′-UTR together with a minimal thymidine kinase (TK) promoter-Renilla reporter gene. 24 h later, promoter activity was measured by a dual luciferase assay. Reporter activity was normalized to a Renilla luciferase internal control and expressed relative to control miRNA inhibitor transfections (set to 1). Data are presented as mean ± S.E. of six samples. Silencing endogenous miR-143 augmented wild type versican 3′-UTR reporter activity but had no effect on mutant. *, p < 0.05. Error bars, S.E.

FIGURE 7.

Myocardin represses versican expression through induction of miR-143. A, 10T1/2 cells were transduced with myocardin or control GFP adenovirus overnight, and miRNA inhibitor for scrambled control or against miR-143 was transfected as indicated. 48 h following transfection, cells were harvested with TRIzol for miRNA reverse transcription, and quantitative PCR was conducted to examine the miR-143 and miR-145 expression. Treatment with miR-143 inhibitor can significantly knock down endogenous and myocardin-mediated induction of miR-143 expression. B–D, versican, myocardin, and SM22α mRNA were measured by qRT-PCR in the myocardin-overexpressed 10T1/2 cells with or without transfection of the miR-143 inhibitor. E and F, versican protein expression was determined by Western blotting (E), and its relative expression was quantified in F. *, p < 0.05. Depletion of miR-143 significantly attenuated myocardin-mediated suppression of versican expression but not on smooth muscle-specific gene SM22α. Error bars, S.E.

FIGURE 8.

PDGF induces versican expression through inhibition of miR-143. Rat primary aortic SMCs were treated with 20 ng/ml PDGF-BB or vehicle for 48 h. Cells were then harvested with TRIzol for total RNA, and qRT-PCRs were performed to measure matrix protein versican and SPP1 (A), smooth muscle gene SM α-actin and myocardin (B), and microRNA miR-143 and miR-145 expression (C). D, qRT-PCR was conducted to examine the versican and SPP1 expression in rat primary SMCs that were PDGF-BB-treated for 48 h following transfection with miR-143 mimic or control mimic overnight. The PDGF-BB treatment-induced relative expression of versican or SPP1 in mimic control transfected cells was set to 1. n = 4. *, p < 0.05. E and F, PDGF-BB-treated rat primary aortic SMCs with or without transfection of mimic miR-143 were harvested for Western blotting with anti-versican antibody. The relative expression of versican was quantified and is plotted in F. The relative expression of versican in vehicle-treated cells was set to 1. *, p < 0.05. Exogenous expression of miR-143 significantly attenuated PDGF-BB-induced versican expression. Error bars, S.E.

FIGURE 9.

Sequential knockdown of miR-143 and versican in PDGF-BB treated SMCs can significantly rescue the depletion of veriscan-mediated inhibition of SMC migration. A, PAC1 SMCs were sequentially transfected with silencing RNA duplex against versican and/or miR-143, as described under “Experimental Procedures” and harvested for qRT-PCR to measure versican mRNA expression. B, following two-round transfection of silencing versican and/or anti-miR-143 RNA duplex, as indicated, PAC1 SMCs were seeded in a Boyden chamber (8-μm pores; BD Biosciences) in serum-free Dulbecco's modified Eagle's medium and then immersed in 10% fetal bovine serum medium with 50 ng/ml PDGF-BB for 5 h. The migrated cells on the bottom surface of the membrane were fixed with 4% paraformaldehyde, stained with 4′,6-diamidino-2-phenylindole (Invitrogen) to visualize nuclei, and counted under fluorescence microscopy. Five identically located fields per membrane were averaged for quantification of migrated cell numbers. p < 0.05. Error bars, S.E.