Bone Morphogenetic Protein 4 Promotes Vascular Smooth Muscle Contractility by Activating MicroRNA-21 (miR-21), which Down-regulates Expression of Family of Dedicator of Cytokinesis (DOCK) Proteins*

Background: miR-21 expression is regulated by BMP4 and plays a critical role in vSMC phenotype regulation. Results: Affinity purification of mRNAs associated with miR-21 yielded nearly all members of the DOCK superfamily. Conclusion: miR-21 targets multiple members of the DOCK superfamily and modulates the activity of Rac1 small GTPase to regulate vSMC phenotype. Significance: This study identified novel targets of miR-21 using a biochemical method. The bone morphogenetic protein 4 (BMP4) signaling pathway plays a critical role in the promotion and maintenance of the contractile phenotype in vascular smooth muscle cell (vSMC). Misexpression or inactivating mutations of the BMP receptor gene can lead to dedifferentiation of vSMC characterized by increased migration and proliferation that is linked to vascular proliferative disorders. Previously we demonstrated that vSMCs increase microRNA-21 (miR-21) biogenesis upon BMP4 treatment, which induces contractile gene expression by targeting programmed cell death 4 (PDCD4). To identify novel targets of miR-21 that are critical for induction of the contractile phenotype by BMP4, biotinylated miR-21 was expressed in vSMCs followed by an affinity purification of mRNAs associated with miR-21. Nearly all members of the dedicator of cytokinesis (DOCK) 180-related protein superfamily were identified as targets of miR-21. Down-regulation of DOCK4, -5, and -7 by miR-21 inhibited cell migration and promoted cytoskeletal organization by modulating an activity of small GTPase. Thus, this study uncovers a regulatory mechanism of the vSMC phenotype by the BMP4-miR-21 axis through DOCK family proteins.

of this effect (8). Upon TGF-␤ or BMP treatment, the R-Smad proteins, which are signal transducers of TGF-␤ and BMPs, associate with primary transcripts of miR-21 (pri-miR-21) in complex with Drosha to promote pri-miR-21 to precursor miR-21 processing and elevated miR-21 expression (8,25). By inducing miR-21, both BMP4 and TGF-␤ lead to knockdown of a negative regulator of contractile gene expression, programmed cell death 4 (PDCD4) (8). We have demonstrated previously that platelet-derived growth factor-BB (PDGF-BB), a potent inducer of the synthetic phenotype in vSMCs, induces miR-221, which in turn promotes (i) down-regulation of contractile genes and (ii) cell proliferation through down-regulation of two target genes, c-kit and p27Kip1, respectively (26). Although down-regulation of PDCD4 by miR-21 is critical for the regulation of vSMC contractile gene expression, it remained unclear whether other effects of TGF-␤ or BMP on vSMC are also mediated by miR-21 and which miR-21 targets are critical mediators of such activities.
There are several approaches to identify targets of miRNAs. The most common uses bioinformatics algorithms to predict potential targets based on the existence of partial complementary MREs in the 3Ј-UTR. Another approach is to identify mRNAs that are down-regulated when a miRNA is overexpressed (8,27,28). miRNA targets can also be enriched by co-immunoprecipitation with exogenously expressed tagged Argonaute (Ago) or GW182 proteins in cells overexpressing miRNA (29,30). Some studies used stable isotope labeling with amino acids in culture to identify proteins that are down-regulated upon overexpression of specific miRNAs (31,32).
In this study, we expressed biotinylated miR-21 in vSMCs and isolated mRNAs that are associated with miR-21 using streptavidin beads followed by microarray analysis for identification of targets. Multiple members of the DOCK family, which are guanine nucleotide exchange factors for Rac and/or Cdc42 (33), were found to be associated with miR-21 in cells. DOCK family genes are not predicted as targets of miR-21 by conventional target prediction algorithms, and little is known about their function in vSMC. Identification of 3Ј-UTR sequences, which are critical for recognition by miR-21, revealed that miR-21 regulates DOCK genes by base pairing at sites other than the seed sequence. We also demonstrate a function for DOCK family members in the regulation of vSMC phenotype by BMP4.

EXPERIMENTAL PROCEDURES
Cell Culture-Human primary pulmonary artery smooth muscle cells (PASMCs) were purchased from Lonza (CC-2581) and maintained in Sm-GM2 medium (Lonza) containing 5% fetal bovine serum (FBS). COS7 and rat pulmonary artery smooth muscle PAC1 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS. Cells were cultured at 37°C in the presence of 5% CO 2 .
Isolation of miR-21 Targets-3Ј-Biotinylated miR-21 mimic (bio-miR-21) and 3Ј-biotinylated control Caenorhabditis elegans miR-67 mimic (bio-cel-miR-67) were synthesized by Dharmacon. PASMCs were transfected with bio-miR-21 or bio-cel-miR-67 mimic at a final concentration of 30 nM using RNAiMax (Invitrogen). Twenty-four hours later, the cells were trypsinized and lysed in TNE buffer (20 mM Tris (pH 7.5), 100 mM KCl, 5 mM MgCl 2 , 0.3% Nonidet P-40, RNase OUT (Invitrogen), protease inhibitor mixture (Roche Applied Science)). The streptavidin-coated magnetic beads (Invitrogen) were incubated with cell lysates at 4°C for 4 h followed by washing with a washing buffer (same as TNE buffer except containing 200 mM KCl). Total mRNAs bound to the streptavidin beads (pulldown RNA) were isolated using TRIzol LS (Invitrogen) according to the manufacturer's instructions. For microarray analysis, isolated mRNAs were subjected to Affymetrix human U133plus 2.0 array at the Clinical Microarray Core, University of California, Los Angeles. Fold enrichment of mRNAs by miR-21 over control cel-miR-67 was calculated.
Quantitative Reverse Transcription-PCR (qRT-PCR)-Total RNAs were isolated using TRIzol (Invitrogen) and reverse transcribed using a first strand cDNA synthesis kit (Invitrogen) according to the manufacturer's instructions. qRT-PCR analysis was performed in triplicates using the SYBR Green Master Mix (Applied Biosystems). The mRNA level was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). For a quantitation of miRNAs, a TaqMan microRNA assay kit (Applied Biosystems) was used according to the manufacturer's instructions, and results were normalized to U6 snRNA. Primers for qRT-PCR analysis are listed in the supplemental information.
Luciferase Reporter Constructs-The full-length 3Ј-UTR sequences were cloned by RT-PCR from mRNAs isolated from PASMCs. Predicted MRE sequences were cloned into the pIS0 vector (Addgene) containing the luciferase gene. Primers for cloning are listed in the supplemental information.
Luciferase Assay-COS7 cells were transfected with luciferase reporter constructs using FuGENE 6 (Roche Applied Science) and a ␤-galactosidase (␤-gal) expression plasmid as an internal transfection control. Twenty-four hours later, cells were transfected using RNAiMax (Invitrogen) with 5 nM miR-21 mimic or control miRNA mimic. Luciferase assays were carried out, and luciferase activities were normalized to ␤-gal activities.
In Vitro Migration Assay-PASMCs transfected with miR-21 mimic, antisense oligonucleotides against miR-21 (anti-miR-21), or siRNAs were plated in 6-well plates, and three scratch wounds were generated with a 200-l disposable pipette tip. Scratch wounds were photographed over 9 h with a Nikon inverted microscope with an attached digital camera, and their widths were quantitated with ImageJ software.
Collagen Matrix Contraction Assay-A collagen matrix contraction assay was performed as described (34). PASMCs transfected with miR-21 mimic, control miRNA mimic, anti-miR-21, or siRNAs were embedded in attached collagen matrices followed by 3 nM BMP4 treatment for 24 h. Twenty-four hours after detachment of the gel from the plate, the gel surface area was quantitated with the ImageJ program.
Detection of Active Rac1-To detect active GTP-bound Rac1, cell lysates of PASMCs transfected with control miRNA mimic or miR-21 mimic were incubated with 20 g of GST-Pak1-PBD (Thermo Scientific), which binds specifically to the GTP-bound form of Rac1, in the presence of glutathione-agarose beads. To measure the total amount of Rac1, Western blot was also performed with anti-Rac1 (Thermo Scientific). The band intensity in the Western blot was quantified, and the levels of Rac1-GTP were normalized to the amount of total Rac1.
Immunoblotting-Cells were lysed in TNE buffer, and total cell lysates were separated by SDS-PAGE, transferred to PVDF membranes (Millipore), immunoblotted with antibodies, and visualized using an enhanced chemiluminescence detection system (Amersham Biosciences). Antibodies used for immunoblotting were: anti-GAPDH antibody (2E3-2E10, Abnova) and anti-human DOCK7 antibody (35). Protein bands were quantitated with the imaging analysis software ImageJ.
Statistical Analysis-The results presented are an average of at least three experiments, each performed in triplicate with standard errors. Statistical analyses were performed by analysis of variance followed by Tukey's multiple comparison test or by Student's t test as appropriate using Prism 4 (GraphPad Software Inc.). p values of Ͻ0.05 were considered significant and are indicated with asterisks.

RESULTS
Identification of Target mRNAs for miR-21-To further understand the physiological role of miR-21 in vSMCs, targets of miR-21 were isolated using a modified method developed previously (36,37). PASMCs were transfected with bio-miR-21 or bio-cel-miR-67 mimic as a negative control. mRNAs associated with bio-miRNA mimic were isolated following affinity purification using streptavidin beads. mRNA targets were identified by comparing isolated pulldown RNAs from cells transfected with bio-miR-21 mimic with those of cells transfected with control mimic by human gene microarray analysis. There are currently 33 genes that are validated as targets of miR-21 according to the miRecords web site. 30 of 33 validated miR-21 targets were enriched more than 2-fold by miR-21 pulldown (supplemental Table S1). None of the housekeeping gene transcripts were enriched (supplemental Table S2). Among previously validated miR-21 targets, E2F1, GLCCI1, and SLC16A10 were not enriched significantly in PASMCs (supplemental Table S1). However, GLCCI1 cannot be found on the microarray, and SLC16A10 is not expressed in PASMCs. Thus, the bio-miR-21 pulldown technique was able to detect all validated targets except one and has a potential to identify novel targets.
Of Ͼ9000 gene transcripts that were enriched by Ն2-fold over control in bio-miR-21 pulldown samples, we examined 26 genes that were not identified previously as miR-21 targets and had the highest enrichment in bio-miR-21 pulldown samples (Ն24-fold over bio-cel-miR-67 pulldown samples) (Fig. 1A). To investigate whether expression of these genes is down-regulated by miR-21, the full-length 3Ј-UTRs or predicted MREs of all 26 genes was inserted at the 3Ј-end of the luciferase gene, and luciferase reporter activity was measured upon overexpression of miR-21 or control cel-miR-67 mimic. As miR-21 MRE or seed sequence is not predicted by target prediction algorithms in the 3Ј-UTR of transcripts encoding PARP9, LGALS14, FGB, DOCK7, SPRYD4, RNF17, and ADAM22, full-length 3Ј-UTRs were examined. For the rest of the genes, predicted miR-21 MREs or seed regions were examined. As a positive control, a miR-21 sensor luciferase construct, which contains two copies of a sequence complementary to miR-21, was used. The miR-21 sensor activity was reduced to 47 Ϯ 6% when miR-21 mimic was expressed (Fig. 1B). Luciferase reporter activities of 22 genes FIGURE 1. Genes enriched by miR-21 pulldown. A, the 26 most highly enriched genes by bio-miR-21 pulldown in PASMCs. The presence of predicted target sites for miR-21 was identified by using PITA and RNA22. Accession indicates NCBI Reference Sequence accession numbers. B, overexpression of miR-21 down-regulates genes that are highly enriched by bio-miR-21 pulldown. Total RNAs were harvested from PASMCs transfected with control or miR-21 mimic, and mRNA levels of the indicated genes relative to GAPDH were measured by qRT-PCR. Fold change of mRNA levels of each gene in cells overexpressing miR-21 mimic in comparison with control (cel-miR-67) mimictransfected cells is presented (black bars). COS7 cells transfected with luciferase construct containing full-length 3Ј-UTR or MRE of the indicated genes were transfected with control (cel-miR-67) or miR-21 mimic. # indicates the luciferase reporter constructs containing a full-length 3Ј-UTR, whereas the rest of the constructs contain only predicted miR-21 MREs. A luciferase vector carrying two copies of a sequence complementary to miR-21 (miR-21 sensor) and a luciferase vector without the 3Ј-UTR sequence (Vector) were used as positive and negative controls, respectively. Luciferase activity was then measured in triplicates. Fold changes by miR-21 mimic transfection in comparison with cel-miR-67 mimic transfection are presented (gray bars). Data represent mean Ϯ S.E. *, p Ͻ 0.01.

Direct Identification of miR-21 Targets in vSMCs
Identification of miR-21 Recognition Sequence in 3Ј-UTR of DOCK4, -5, and -7 Transcripts-DOCK4, -5, and -7 are known as guanine nucleotide exchange factors for Rac1 GTPases and play a role in cell migration and cytoskeletal organization in neuronal and tumor cells (33,38). DOCK4, -5, and -7 were not predicted as miR-21 targets by the algorithms whose prediction is based on a seed-matched sequence and cross-species conservation of the seed sequence. RNA22, which predicts targets based on a pattern-based method (39), found nine potential MREs in the 3Ј-UTR of DOCK4 that are partially complementary to the miR-21 sequence (Fig. 3, A and B). To determine whether any of these predicted MREs are responsible for knockdown by miR-21, luciferase reporters containing individual MREs were transfected into COS7 cells together with miR-21 or control cel-miR-67 mimic. miR-21 significantly reduced the luciferase activity of MRE1 (43 Ϯ 1%) and MRE6 (58 Ϯ 1%) (Fig. 3A). Mutations in MRE1 or MRE6 (Fig. 3B, mt), which disrupted critical base pairing with miR-21, abrogated the inhibition of luciferase activity by miR-21 (Fig. 3C), suggesting that MRE1 and MRE6 are critical target sites for recognition of DOCK4 mRNAs by miR-21.
Down-regulation of DOCKs by miR-21 Inhibits Rac1 Activity and Functions-As DOCK4, -5 and -7 are linked to the activation of the Rac1 GTPase (33, 38), we hypothesized that miR-21 might modulate vSMC migration and cytoskeletal organization by inhibiting Rac1 activity. To examine whether miR-21 modulates Rac1 GDP/GTP exchange activity in vSMCs, the amount of active Rac1 was measured by pulldown assays using the GST-Pak1-PBD domain fusion protein, which specifically binds active Rac1. Treatment with PDGF-BB, which is known to activate Rac1, was included as a positive control and triggered a ϳ3-fold increase in active Rac1 (Fig. 5A). miR-21 reduced the level of active Rac1 by ϳ50% compared with control cel-miR-

Direct Identification of miR-21 Targets in vSMCs
67-transfected cells without affecting the amount of total Rac1 (Fig. 5A), suggesting that elevation of miR-21 decreases active GTP-bound Rac1. Next, we examined a role for miR-21 in cell migration by performing a scratch wound assay (34). To this end, a wound was created in a monolayer of PASMCs, and images were captured at the beginning and at regular intervals during cell migration to close the wound. Similarly to the cells treated with BMP4 (Fig. 5B, green line), the migration distance was reduced to 47 Ϯ 3% 9 h after a scratch wound was generated in miR-21-transfected cells (Fig. 5B, red line) compared with control miRNA-transfected cells (Fig. 5B, black line). In contrast with the miR-21 mimic, transfection of anti-miR-21 increased cell migration (Fig. 5B, magenta line), indicating that a differential level of expression of miR-21 determines vSMC migration. When DOCK4, -5, and -7 were down-regulated individually or simultaneously by siRNAs by 54 Ϯ 3, 59 Ϯ 15, and 64 Ϯ 4%, respectively (supplemental Fig. S1A), cell migration was significantly reduced comparably with that of miR-21overexpressing cells (Fig. 5C), suggesting that DOCK4, -5, and -7 regulate vSMC motility. This result also indicates a redundant function among DOCK4, -5, and -7 for the regulation of cell migration. When PASMCs were treated with NSC23766, an inhibitor of Rac1 GDP/GTP exchange activity (38), cell migration was reduced similarly to that in DOCK4, -5, and -7 knockdown cells (Fig. 5D). Furthermore, exogenous expression of DOCK7 mRNAs (DOCK7 wo3Ј-UTR ), which have the 3Ј-UTR deleted and are resistant to miR-21, in rat pulmonary artery smooth muscle PAC1 cells blocked BMP4-induced inhibition of migration (Fig. 5E, green square versus blue triangle). Thus, regulation of DOCK4, -5, and -7 levels is critical for the migratory function in vSMCs. Altogether, these results indicate that the GDP/GTP exchange activity of Rac1 is critical for vSMC migration, and the BMP4-miR-21 axis suppresses vSMC migration by down-regulation of DOCK proteins.
Next, in vitro collagen gel contraction assays were performed in PASMCs transfected with miR-21 mimic, anti-miR-21, or siDOCKs to evaluate the effect of miR-21-mediated down-regulation of DOCKs in the cytoskeletal organization of PASMCs. Similarly to BMP4-treated cells, transfection of miR-21 mimic reduced gel size by ϳ20% compared with control samples (Fig.  5H). Overexpression of miR-21 mimicked the effect of BMP4 and induced contraction, whereas transfection of anti-miR-21 abolished BMP4-induced contraction (Fig. 5H), indicating a critical role of miR-21 expression in contraction of the collagen gel. Down-regulation of DOCK4 or DOCK5 by siRNA also reduced the size of collagen lattice by 18 Ϯ 3 and 20 Ϯ 4%, respectively (Fig. 5H), which is similar to that of miR-21-transfected cells, suggesting that down-regulation of DOCK4 or DOCK5 by miR-21 is sufficient to mediate vSMC contraction. Knockdown of DOCK7 showed no effect on contraction (Fig.  5H). Consistently, knockdown of DOCK4 or DOCK5 elevated the levels of contractile genes (ASMA and CNN), whereas siDOCK7 had no effect (Fig. 5I), suggesting a specific role of DOCK4 and -5 in the regulation of contractile gene expression and contractility. Treatment of cells with the Rac1 inhibitor NSC23766 did not affect the contraction of the collagen lattice (supplemental Fig. S2). Thus, DOCK4 and DOCK5 promote cytoskeletal remodeling in a Rac1-independent manner. These results indicate that miR-21 modulates cell migration and contractility through down-regulation of DOCK family members in vSMCs. Therefore, the miR-21-DOCK axis regulates critical characteristics of the vSMC phenotype, such as motility and cytoskeletal organization, in response to BMP4 and PDGF signaling (Fig. 6).

DISCUSSION
In this study, we identified the DOCK family members as miR-21 targets downstream of BMP4 and provided evidence that their down-regulation by miR-21 is critical for promoting the contractile vSMC phenotype through inhibiting cell migration and promoting contractility (Fig. 6). The role of DOCK proteins in the regulation of cell motility, cytoskeletal organization, phagocytosis, and polarity has been elucidated in various cell types, especially in the nervous system (33). However, Immunoblotting of total cell lysates with anti-Rac1 antibody is shown to demonstrate total amount of Rac1 (top panel, Total Rac1). The level of Rac1-GTP relative to total Rac1 is presented as a relative Rac1 activity (bottom panel). B, PASMCs transfected with control cel-miR-67 mimic (Control; black diamonds), miR-21 mimic (miR-21; red circles), or anti-miR-21 (magenta triangles) were subjected to the scratch wound assay. As a control, PASMCs were treated with 3 nM BMP4 (green squares). The distance of migration was measured using ImageJ over 9 h after a scratch wound was introduced. C, PASMCs transfected with 25 nM non-targeting control siRNA (siControl); siRNA against DOCK4 (siDOCK4), DOCK5 (siDOCK5), or DOCK7 (siDOCK7); or miR-21 mimic (miR-21) were subjected to the scratch wound assay. D, PASMCs transfected with siControl or siDOCK4 were subjected to the scratch wound migration assay in the presence or absence of 50 M Rac1 inhibitor (NSC23766). E, rat PASMC PAC1 cells were transfected with control vector (Control) or DOCK7 expression construct carrying the DOCK7 cDNA deleted in the 3Ј-UTR (DOCK7 wo3Ј-UTR ) followed by the scratch wound assay in the presence or absence of 3 nM BMP4. F, levels of DOCK4, -5, or -7 mRNA relative to GAPDH or miR-21 relative to U6 snRNA were measured by qRT-PCR in PASMCs stimulated with 20 ng/ml PDGF-BB or 3 nM BMP4 for 24 h. Data represent mean Ϯ S.E. *, p Ͻ 0.0001; **, p Ͻ 0.01; ***, p Ͻ 0.001. G, PASMCs transfected with siRNA (siControl, siDOCK4, siDOCK5, or siDOCK7) were subjected to the scratch wound assay in the presence or absence of 20 ng/ml PDGF-BB. Data represent mean Ϯ S.E. H, PASMCs were transfected with siControl, siDOCK4, siDOCK5, siDOCK7, miR-21 mimic, or anti-miR-21 followed by stimulation with 3 nM BMP4 or no stimulation (None) for 24 h. The PASMC embedded collagen lattices were photographed by using a digital camera to measure gel contraction. The relative size of collagen gel was quantitated using ImageJ. Data represent the mean percentage of the collagen gel shrinkage compared with unstimulated control. I, levels of contractile genes. ␣-Smooth muscle actin (ASMA) and calponin1 (CNN) mRNA levels relative to GAPDH were measured by qRT-PCR in PASMCs transfected with siControl (siCont), siDOCK4, siDOCK5, or siDOCK7. Data represent mean Ϯ S.E. *, p Ͻ 0.01. FEBRUARY 3, 2012 • VOLUME 287 • NUMBER 6 no study on DOCK proteins has been performed in vascular cells previously. We previously reported that DOCK7 protein is asymmetrically distributed in unpolarized hippocampal neurons and becomes enriched in axons during the development of this process (35). However, the mechanism underlying the regulation of DOCK7 in hippocampal neurons is unclear. It is interesting to speculate that such specific localization of DOCK7 might be due to selective expression of miR-21.

Direct Identification of miR-21 Targets in vSMCs
Using a miRNA pulldown method, we were able to identify DOCK family genes as targets of miR-21. Over 29,000 genes are predicted as targets of miR-21 by at least one of 11 commonly used target prediction algorithms according to the miRecords web site. Thus, experimental methods to purify/identify targets of specific miRNAs are needed. In vivo study of targets of several miRNAs, including miR-1, miR-16, miR-30a, miR-155, and let-7b, using pulsed stable isotope labeling with amino acids in culture analyses in HeLa cells that overexpress these miRNAs indicates that roughly 300 proteins are down-regulated by at least 30% (32). The same study estimated that about 60% of the 300 down-regulated proteins contain seed-matched sites (Watson-Crick consecutive base pairing between mRNAs and the miRNA at positions 2-7 counted from the 5Ј-end of the miRNA) in the 3Ј-UTR regions of transcripts (32). Among the 33 previously validated miR-21 targets, only eight targets are predicted by a seed sequence-based algorithm. Non-seedbased algorithms tend to predict too many sites (on average ϳ3,000 -11,000 MREs per miRNA), which defeats the purpose of using target prediction algorithms. Thus, the biochemical enrichment of miRNA targets presented in this study is useful. We identified ϳ26 genes that are Ն24-fold enriched in miR-21 pulldowns over control cel-miR-67 in PASMCs. We tested 28 predicted MREs located in the 3Ј-UTR of mRNAs associated with bio-miR-21, including 16 predicted MREs or sequences partially complementary to miR-21 localized in the 3Ј-UTR of DOCK4, -5, or -7 transcripts. Independently, miR-34a target analysis was performed in HCT116 and K562 cells using the bio-miR-34a pulldown method and confirmed that genes enriched in bio-miR-34a pulldowns by as little as 2.5-fold over control miRNA pulldowns in both cells have 3Ј-UTRs that are regulated directly by miR-34a as measured by the luciferase reporter activity assay (40). Thus, additional miR-21-regulated genes are likely, and further verification and investigation of genes that are enriched in bio-miR-21 pulldowns in PASMC are warranted to understand the biological functions of miR-21 in vSMCs.
Six of the 22 miR-21-associated mRNAs that are enriched Ͼ24-fold over control miRNAs (PARP9, DOCK7, ADAM22, RNF17, LGALS14, and SPRYD4) do not contain miR-21 seed sequence or predicted MREs. RNAhybrid predicted some of the DOCK genes; however, this algorithm predicts on average ϳ11,000 target sites per miRNA. MRE sites in DOCK4, DOCK5, and DOCK7 3Ј-UTRs identified in this study contain complementary sequences of 6 -11-mers starting somewhere between position 1 and 15. These results suggest that base pairing between a miRNA and target mRNA can be important downstream of the seed site.
It is intriguing that miR-21 targets multiple transcripts belonging to the same family. There are a few other examples of a single miRNA targeting multiple genes of the same family. miR-200 targets the zinc finger homeodomain enhancer-binding protein (ZEB) family of transcription repressors; ZEB1 and ZEB2 regulate the epithelial-to-mesenchymal transition (41). miR-164 targets the NAC domain transcription factors CUP-SHAPED COTYLEDON1 (CUC1) and CUC2 that establish axillary meristems of leaves in plant (42). In both cases, products of the same gene family share a redundant function as in the case of DOCK4, -5, and -7 and Rac1 regulation in the regulation of cell migration (33). Although examples of a single miRNA targeting multiple genes in the same signaling pathway or with related biological functions have been reported previously, our results are striking because they suggest that a single miRNA targets multiple transcripts of the same gene family. In the case of miR-21 MREs identified in the 3Ј-UTR of human DOCK7 (MRE1), an 8-mer at the 5Ј-end is 100% conserved in mouse. For human DOCK4, a 6-mer complementary sequence starting at position 3 in MRE1 is also 100% conserved in mouse, whereas in MRE6, only six of the 11 residues complementary to miR-21 starting at position 3 are conserved in the mouse 3Ј-UTR sequence. However, two MREs found in DOCK5 are not conserved in mouse, suggesting that only some MREs are evolutionally conserved.
Increased proliferation and migration of vSMCs are associated with various stimuli, including activation of PDGF signaling, inhibition of BMP/TGF␤ or Notch signaling, hypoxia treatment, mechanical stretch, and activation of Rac1 (43,44). However, a role for DOCK4, -5, or -7 has not been characterized in vSMCs previously. Our study illustrates both common and distinct roles of DOCK family of proteins in the regulation of vSMC phenotype (Fig. 6). DOCK4 is known to mediate cell migration promoted by the PDGF signal in NIH3T3 cells (45). In vSMCs, BMP and PDGF signals antagonize each other and modulate vSMC phenotype during vascular injury repair or pathogenesis of vascular proliferative diseases (46). Thus, the BMP4-miR-21-DOCK4 axis is a novel mechanism of antagonism between BMP and PDGF signaling pathways in addition to the miR-24-Trb3 axis, which has been described previously (34). Interestingly, cytoskeletal remodeling is mediated by DOCK4 and DOCK5, but not by DOCK7, through a mechanism that is independent of Rac1. As DOCK4 and -5 contain multiple Src homology 3 domains that are not found in DOCK7 (33), we speculate that the Src homology 3 domain might play a critical role in the cytoskeletal remodeling through docking other signaling molecule(s). miR-21 is elevated in most human cancers and has been shown to function as an oncogene by promoting cell growth and migration (17). Indeed, many validated targets of miR-21 are tumor suppressors, including PDCD4 (47), PTEN (48), and tropomyosin 1 (TPM1) (21). Homozygous deletion of DOCK4 gene occurs during tumor progression in the TP53-NF2 mouse model and is associated with increased invasion of tumor cells (49). Furthermore, a missense mutation (Pro-1718 to Leu) of DOCK4 that results in defective activation of the Rap1 GTPase (49) has been identified in human prostate and ovarian cancers. Thus, in this context, DOCK4 is a novel target of miR-21 with tumor suppressor function. It is intriguing, however, that miR-21, which promotes invasion and migration of cancer cells (50), inhibits migration in vSMCs. This observation is indicative of a cell type-specific function of miR-21. A potential mechanism of the cell type-specific function of miRNAs involves how different sets of genes are targeted by a miRNA due to (i) a cell typespecific expression of mRNA targets and/or (ii) a cell type-specific recognition/targeting of mRNA targets by a miRNA. A potential advantage of the miRNA target pulldown approach is that it allows identification of miRNA targets in a context-dependent manner. In addition to the relevance to vSMC biology, further investigation of the targets identified by the miR-21 pulldown may provide insights for understanding the sequence requirements for specific recognition of mRNA targets by miRNA and facilitate the development of more accurate target prediction algorithms.