miR-1-Mediated Induction of Cardiogenesis in Mesenchymal Stem Cells via Downregulation of Hes-1

MicroRNAs (miRNAs, miRs) have the potential to control stem cells fate decisions. The cardiac- and skeletal-muscle-specific miRNA, miR-1, can regulate embryonic stem cells differentiation to cardiac lineage by suppressing gene expression of alternative lineages. Accordingly, we hypothesized that overexpression of miR-1 may also promote cardiac gene expression in mesenchymal stem cells. Since Notch signaling could inhibit muscle differentiation, a process in contrast with the effect of miR-1, miR-1-mediated repression of Notch signaling may contribute to the observed effects of miR-1 in mesenchymal stem cells. Thus, mesenchymal stem cells were infected by lentiviral vectors carrying miR-1, and cells expressing miR-1 were selected. Alterations in Notch signaling and cardiomyocyte markers, Nkx2.5, GATA-4, cTnT, and CX43, were identified by Western blot in the infected cells on days 1, 7, and 14. Our study showed that the downstream target molecule of Notch pathway, Hes-1, was obviously decreased in mesenchymal stem cells modified with miR-1, and overexpression of miR-1 promotes the specific cardiac gene expression in the infected cells. Knockdown of Hes-1 leads to the same effects on cell lineage decisions. Our results indicated that miR-1 promotes the differentiation of MSCs into cardiac lineage in part due to negative regulation of Hes-1.


Introduction
Stem cell transplantation has been extensively investigated as a therapy to regenerate cardiac tissue aer myocardial infarction. Mesenchymal stem cells (MSCs) can be easily isolated and expanded, and they possess neurogenic, chondrogenic, adipogenic, osteogenic, and myogenic properties under spe-ci�c differentiating conditions [1,2]. Furthermore, these cells have a stable genetic background and low risk of immune rejection. As a result, they are oen used as seeding cells in tissue engineering and stem cell therapy. Precise regulation of cell fate decisions is a prerequisite for future therapeutic use of MSCs. Transcription factors that regulate pluripotency or lineage-speci�c gene and protein expression have been a major focus of stem cells research. Numerous signaling pathways, including Wnt, BMP, and Notch signaling pathways, regulate cell fate decisions during MSC differentiation and can be utilized to in�uence lineage choices in vitro [3][4][5]. Notch signaling plays an essential role in a variety of biological processes, including cell differentiation, cell fate speci�cation, and patterning during embryonic and postnatal development [6]. In mammals, the Notch signaling pathway is comprised of �ve transmembrane ligands: deltalike-(Dll-) 1, (Dll-)3, (Dll-)4, Jagged (Jag-1), and Jagged (Jag-2), four transmembrane receptors: Notch-1, -2, -3, and -4, and downstream target genes, such as bHLH (basic helixloop-helix) proteins: Hes (hairy/enhancer of split) and Hey (Hes-related protein). e pathway is crucial for cell-tocell interaction during cardiovascular development and may in�uence cardiac differentiation, proliferation, and apoptotic events [7][8][9].
In addition to the numerous transcription factors and signaling molecules that control development of cardiac cells [10], microRNAs (miRNAs, miRs) also play critical roles in cardiac differentiation [11][12][13]. ese small noncoding RNAs are naturally occurring molecules that are transcribed in the nucleus, oen under the control of speci�c enhancers. ey are processed by the RNAses Drosha/DGCR8 and Dicer into mature ∼22 nucleotide RNAs, which can repress target mRNA expression by binding to miRNA regulatory elements (MREs) via Watson-Crick base pairing between the miRNA "seed region" and sequences commonly located in the 3 ′ -untranslated region (3 ′ -UTR) of target mRNAs [11,[14][15][16]. Previous studies have revealed that miRNAs play an important role in various cellular processes, including proliferation, differentiation, apoptosis, and development [17][18][19]. Some of these small RNAs are expressed in a lineage-speci�c fashion and, thus, have the potential to control stem cell fate decisions [20,21]. Further studies have demonstrated that miRNAs contribute to the regulation of various signaling pathways via the repression of target genes, which results in the regulation and modulation of signal transduction [22]. MiR-1, a cardiac-and skeletal-muscle-speci�c miRNA, is involved in muscle differentiation and maintenance of muscle gene expression in both mammals and �ies [11][12][13]. Notch signaling promotes neural differentiation and inhibits muscle differentiation in embryonic stem cells (ESCs) [23,24], effects that are opposite to those of miR-1. Previous work by Kwon et al. showed that miR-1 directly represses the Notch ligand delta in Drosophila [12]. ree orthologs of Drosophila delta have been identi�ed in mice: Dll-1, Dll-3, and Dll-4. Recently, Ivey and his colleagues demonstrated a new role for miRNAs in regulating ESCs differentiation. ey found that miR-1 directs mesoderm formation from ESCs and regulates differentiation to cardiac lineage by suppressing gene expression of alternative lineages. is effect is partly due to miR-1 directly repressing the Notch ligand Dll-1 [25]. Accordingly, we hypothesized that overexpression of miR-1 may lead to similar effects in the culture MSCs.

Animal Ethics.
All procedures were performed in accordance with guidelines of Laboratory Animal Care formulated by the National Society for Medical Research and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication no. 85-23, revised 1996).

Primary
Culture and Characterization of MSCs. 6-weekold male C57BL/6 mice were sacri�ced by cervical dislocation. e hind legs and vertebrae were dissected and carefully separated from adherent tissues. Aer the tips of each bone were removed, bone marrow was collected by �ushing out the content of femurs and tibias with phosphate buffered saline (PBS). e collected cells were cultured in complete medium, consisting of Dulbecco's Modi�ed Eagle's Medium with Nutrient Mixture F12 (DMEM/F12; GIBCO), 10% fetal bovine serum (GIBCO), and 1% penicillin/streptomycin (GIBCO). e cultured cells were analyzed by �uorescenceactivated cell sorting (FACS) as previously described [26]. e differentiation of MSCs in vitro towards the adipogenic and the osteogenic lineage is shown as previously described [27,28].

Recombinant Lentiviral Vectors Construction, Cells Infection, and Stable Cell Line Generation.
To produce recombinant lentiviral vectors encoding miR-1, mature miR-1, TRE promoter, and enhanced green �uorescent protein (eGFP) sequences were inserted into plasmids to produce pUp-TRE, pDown-miR-1, and pTail-IRES/eGFP; scramble sequence was set as negative control. pLV.EX3d.P/puro-TRE > miR-1 > IRES/eGFP was obtained with incubation of donors and accepter vectors catalyzed by LR Clonase (Gateway LR Clonase Plus Enzyme Mix, Invitrogen). Plasmid was then sequenced and puri�ed for lentivirus envelope. Envelope helper plasmids: pLV/helper-SL3, pLV/helper-SL4, and pLV/helper-SL5, with pLV. EX3d.P/puro-TRE-miR-1-IRES/eGFP or pLVrtTA/neo which contains the imperative elements for virus packaging, were cotransfected into 293T cells with lipofectamine 2000, according to the manufacturer's instructions (Invitrogen) for generation of Lenti-miR-1-eGFP/puro or Lenti-rtTA/neo, respectively. To perform lentiviral infections, MSCs were �rstly treated by Lenti-rtTA/neo; 48 h later, infected cell populations were selected in 0.5 mg/mL neomycin and refresh medium every two days. Selection was terminated when control cells were completely dead and antibiotic free medium was used for propagation. Neomycin resistant cells were infected by Lenti-miR-1-Puro/GFP and grown with 2 g/mL puromycin. Double resistance cells were then ultimately obtained, and 2 g/mL doxycycline was added to medium and intrigue expression of miR-1. MSCs infected with miR-1 recombinant lentiviral vectors carrying GFP were named MSCs miR-1 , and MSCs infected with mock lentiviral vectors carrying GFP were named MSCs , which used to determine whether the mock vectors without carrying miR-1 could in�uence the molecules of cells signaling pathway or cell lineage decisions compared to the control cells-MSCs.
To determine whether repression of Hes-1 could account for a subset of the effects of miR-1 on cell lineage decisions, we used the same way to construct lentiviral vectors carrying Hes-1 to transfect MSCs. To produce recombinant lentiviral vectors encoding Hes-1-shRNA, three types of plasmids: pAJ-U6-shRNA-CMV-Puro/GFP, psPAX2 (gag/pol element), and pMD2.G (VSVG element) were transfected into 293T cells according to the instructions. Aer 48 h transduction, infected MSCs were selected with 2 g/mL puromycin until mock-transfected cells died and were maintained under selection pressure throughout the generation of stable Hes-1-shRNA line. Cells transfected with Hes-1-shRNA recombinant lentiviral vectors were named MSCs Hes-1-shRNA . Stable shRNA line was cultured in a 5% CO 2 -humidi�ed incubator at 37 ∘ C.

Quantitative Real-Time PCR (qRT-PCR) Identify the Efficiency of Gene Transfection.
Total RNA was extracted from each sample with Trizol reagent (Invitrogen) according to the manufacturer's instructions. Expression of miR-1 was detected by qRT-PCR using All-in-One miRNA qRT-PCR Detection Kit (GeneCopoeia) and All-in-One miRNA qPCR Primer (GeneCopoeia). e primer sequence is 5 ′ -CAGTCT-GGCGAGAGAGTTCC-3 ′ . e levels of miR-1 transcripts were normalized to the control U6 mRNA, which primer sequence was 5 ′ -TCGTGAAGCGTTCCATATTTTTAA-3 ′ .
To test the expression of Hes-1 in the transfected cells, 2 g of total RNA extracted from each sample was reversed transcribed into �rst-strand cDNA by RevertAid First Strand cDNA Synthesis Kit (Fermentas), according to the manufacturer's instructions. e synthesized cDNA was used for real-time quantitative PCR analysis of Hes-1 mRNA expression with SYBR Premix Ex Taq (TaKaRa). e sense sequence of Hes-1 primers was 5 ′ -AGAAGAGGCGAA-GGGCAAGA-3 ′ , whereas the antisense sequence was 5 ′ -CGGAGGTGCTTCACAGTCAT-3 ′ . Expression levels were quanti�ed by normalizing the values relative to the mouse housekeeping gene: -actin content, which primer sequence was 5 ′ -CAGCCTTCCTTCTTGGGTAT-3 ′ , and the antisense sequence was 5 ′ -TGGCATAGAGGTCTTTACGG-3 ′ . Relative gene expression was calculated by the 2 −ΔΔCT method [29].

Statistical
Analysis. Data were presented as mean values and standard deviation. A method of ANOVA (analysis of variance) with Scheffe's post hoc test was used to identify differences among all groups. P values of less than 0.05 were considered to be statistically signi�cant.

Phenotypic Characterization and Differentiation
Capacity of Cells. Cells were scattered in a number of colony distributions 3 days aer planting. At days 8-9, the bottle was covered with long spindle-like cells. e passaged cells (mostly �broblast-like cells) were uniformly distributed and covered the bottom every 4-5 days. e 3rd passage of cells highly expressed the MSCs surface marker molecules, CD29 and CD90, and negatively expressed the blood cell surface molecules, c-kit, CD34, and CD45. ese cells were differentiated in vitro using adipogenic and osteogenic induction media. Following 3 weeks of adipogenic induction, the cells stained positive for Oil Red O, showing a lipidladen adipocyte phenotype. Similarly, when induced with osteogenic induction medium for 3 weeks, these cells showed osteogenesis upon staining with Von Kossa for calcium deposits. ese results demonstrated that the stem cells possess pluripotent of differentiation ability.

Efficiency of Gene Transfection and miR-1 Expression.
Aer infection with miR-1 recombinant lentiviral vectors, MSCs overexpressed GFP (Figures 1(a) and 1(b)). Quantitative real-time PCR test indicated that the efficiency of gene transfection in the MSCs miR-1 group was similar to that in the MSCs group (91.2% versus 90.3%). e expression of miR-1 in the MSCs miR-1 group was 212-fold higher than that in the MSCs group ( 1 and 226-fold higher than expression in MSCs group ( 1 (Figure 1(c)).
e passaged MSCs grew as �broblast-like or long spindle-shaped (Figure 3(a)). Twenty-four hour aer transfected with miR-1, the stem cells had no change in appearance (Figure 3(b)). Seven days aer modi�ed with miR-1, the cells assume star or spindle-shaped, with fewer pseudopodia (Figure 3(c)). On day 14, these infected cells appearance of polygonal or short spindle-shaped most looks like cardiomyocytes (Figure 3(d)). Intercalated disc in a little cells was detected by using electron microscope. However, even though overexpression of miR-1 could promote cardiac gene expression in MSCs and most infected cells appearance of cardiomyocyte-like two weeks later, we did not detect any beating cardiac cells during the differentiation process. Maybe MSCs differentiate into cardiomyocytes needed spe-ci�c conditions besides miR-1.

Knockdown of Hes-1 Promotes the Expression of Cardiac Makers in MSCs.
To study whether downregulation of Hes-1 protein by miR-1 could account for a subset of the effects of miR-1 on cell lineage decisions, we used shRNA constructs directed against distinct regions of Hes-1 to generate Hes-1-shRNA cell line. In our experiment, the Hes-1 mRNA level was about 69% lower in MSCs Hes-1-shRNA than in a control line expressing a scrambled shRNA construct. By using Western blot methods, we show that the expression of Hes-1 is signi�cantly decreased in MSCs Hes-1-shRNA on days 7 and 14. Nkx2.5 and GATA-4 expression were induced in MSCs Hes-1-shRNA on day 7, but they were not detected in MSCs. Furthermore, cTnT and CX43 were expressed on day 7 and at even higher levels on day 14 in MSCs Hes-1-shRNA cells. Both cTnT and CX43 were negative for expression in controls. Although the effect of Hes-1 knockdown on expression of Nkx2.5, GATA-4, cTnT, and CX43 was not as robust as the expression in MSCs miR-1 , the trends were similar.

Discussion
MSCs have been used to regenerate cardiac tissues by virtue of their capability to transdifferentiate into cardiomyocytes. Improving the survival and differentiation of transplanted cells in the infarction site is critical for improving the efficiency of stem cell therapies. Notch signaling pathway is involved in many differentiation processes and lineage decisions in fetal and postnatal development. Signaling is initiated via ligand-receptor interactions on neighboring cells. e interaction leads to a series of successive proteolytic cleavages. First, extracellular cleavage of Notch occurs by TACE (TNF (tumour necrosis factor -) converting enzyme). is is followed by transmembrane cleavage bysecretase, releasing the NICD (Notch intracellular domain), which then translocates to the nucleus and heterodimerizes with the transcriptional regulator RBP-J (recombinant signal-binding protein 1 for J ), converting it from a transcriptional repressor to an activator. NICD binding  to RBP-J replaces the corepressor transcriptional complex with a coactivator complex, which in turn triggers the transcription of Notch downstream target genes, such as bHLH proteins: Hes and Hey [30,31]. Notch signaling has been shown to direct cells toward alternate differentiation fates [32]. e contribution of Notch signaling in regulation of cardiac marker gene expression has been supported by various reports demonstrating a Notch-mediated suppression of cardiomyogenesis in Xenopus, Drosophila, and ESCs [23,33,34]. Modulation of Notch-1 signaling in MSCs may improve MSCs function in terms of mobilization or recruitment to the injured heart [9]. It has been shown that Notch signaling in�uences the cell fate decision between mesodermal and neuroectodermal cell fates during ESCs differentiation and downregulation of Notch-1 signaling could induce cardiogenesis in ESCs [23]. Our study demonstrated that the expression of Notch molecules: Notch-1, Notch-2, Notch-4, Dll-1, Dll-4, Jag-1, Hes-1, and Hey-1 were detected in MSCs. Knockdown of Hes-1 contributes to the induction of early cardiac makers, Nkx2.5 and GATA-4, and cardiomyocyte-speci�c makers, cTnT and C�43 in MSCs. Basing on this �nding, one may speculate that Hes-1 plays a critical role in promoting the expression of cardiac gene in the stem cells. MiRNAs regulate gene expression and act as important factors in the regulation of stem cell function [35][36][37][38]. ey are also involved in controlling cell fate, and it is likely that they regulate these decisions by regulating numerous genes and pathways. MiR-1, a muscle-speci�c miRNA, has been suggested to play a role in cardiogenesis and cardiac gene expression [11,12]. e study of Kwon et al. had previously shown in Drosophila that miR-1 directly targets the Notch ligand delta for repression [12]. In our study, two orthologs of Drosophila delta were identi�ed in mouse MSCs-Dll-1 and Dll-4. Overexpression of miR-1 promotes differentiation of MSCs into the cardiac lineage. However, our semiquantitative data showed that the Notch ligand delta did not alter during the MSCs miR-1 differentiation process (on days 1, 7, and 14). In the meanwhile, other Notch upstream molecules, Notch-1, -2, -4, and Jag-1, did not change in the MSCs miR-1 at the same time points. ese results indicated that miR-1 does not in�uence the Notch upstream molecules of mouse MSCs. Interestingly, expression of Hes-1 was decreased in MSCs miR-1 on days 7 and 14. �nd speci�c knockdown of Hes-1 can lead to similar cell fate trends as miR-1 overexpression in MSCs. ese results were similar to the recent reports which indicated that Notch-1 inhibition promotes cardiac differentiation [23,24] and the experiment of Ivey et al. that miR-1 regulates ESCs differentiation to cardiac lineage due to miR-1 inhibiting Dll-1 [25]. Yet our study demonstrated that it is the Notch downstream target molecule, Hes-1, but not Notch-1 or Dll-1, which directly contributes to the induction of cardiogenesis in the mouse MSCs. MiR-1 promotes the differentiation of MSCs into the cardiac lineage due to negatively regulation of Hes-1.
is effect might depend on miR-1 directly repressing Hes-1. However, as previously described, Notch signaling is initiated via ligand-Notch receptor interactions on neighboring cells. e interaction leads to a series of successive proteolytic cleavages, which in turn triggers the transcription of Notch downstream target genes. Since miR-1 does not alter Notch ligands and receptors during the differentiation process of MSCs miR-1 , one may speculate that the Notch signaling system may crosstalk with others signaling pathways such as Wnt, BMP, and TNF-, which have been well characterized as enhancing the expression of cardiac markers in stem cells [3,5,39,40]. Maybe miR-1-mediated induction of cardiogenesis in MSCs due to regulation of other signaling pathways, thereby negatively in�uence Hes-1. Even though overexpression of miR-1 could promote cardiac gene expression in MSCs, we did not detect any beating cardiac cells during the differentiation process of cultured MSCs modi�ed with miR-1. One possibility is that MSCs differentiate into cardiomyocytes only under speci�c conditions, such as myocardial microenvironment. Maybe miR-1 only play a partial role in or accelerate the differentiation process.
Growing evidence demonstrate that bone marrowderived MSCs have been proposed as a novel therapeutic approach for improvement of infracted heart function through regeneration of myocardium. However, low survival and differentiation rate of transplanted cells in ischemic myocardium in�uence the outcome of stem cell transplantation for treatment of the ischemic disease. Cell sheet gras with genetically engineered properties to promote the differentiating into the cardiac lineage may offer a potential approach to repair dead or injured myocardium. Whether the survival and differentiation of transplanted miR-1 modi�ed cells to the infarction site would improve the efficiency of stem cell therapies needs further investigation.

Conclusion
In conclusion, our results indicate that the muscle-speci�c miRNA, miR-1, promotes the differentiation of MSCs into the cardiac lineage by repression of Hes-1. MiRNAs may offer a means to direct the differentiation of MSCs into desired fates. MSCs genetically modi�ed with miR-1 could signi�cantly advance the efficacy of stem cell differentiation.