Involment of RAS/ERK1/2 signaling and MEF2C in miR-155-3p inhibition-triggered cardiomyocyte differentiation of embryonic stem cell

MicroRNAs (miRNAs) are short, noncoding RNAs that regulate post-transcriptional gene expression by targeting messenger RNAs (mRNAs) for cleavage or translational repression. Growing evidence indicates that miR-155 expression changes with the development of heart and plays an important role in heart physiopathology. However, the role of miR-155 in cardiac cells differentiation is unclear. Using the well-established embryonic stem cell (ESC), we demonstrated that miR-155-3p expression was down-regulated during cardiogenesis from mouse ESC. By contrast, the myogenic enhance factor 2C (MEF2C), a predicted target gene of miR-155-3p, was up-regulated. We further demonstrated that miR-155-3p inhibition increased the percentage of embryoid bodies (EB) beating and up-regulated the expression of cardiac specific markers, GATA4, Nkx2.5, and cTnT mRNA and protein. Notably, miR-155-3p inhibition caused upregulation of MEF2C, KRAS and ERK1/2. ERK1/2 inhibitor, PD98059 significantly decreased the expression of MEF2C protein. These findings indicate that miR-155-3p inhibition promotes cardiogenesis, and its mechanisms are involved in the RAS-ERK1/2 signaling and MEF2C.


INTRODUCTION
Heart disease, including myocardial infarction, is the leading cause of death worldwide.Myocardial infarction causes irreversible damage to the myocardium due to the limited capacity of postnatal hearts to generate new cardiomyocytes.It is identified that cardiomyocytes lost by apoptosis are renewed at a rate of approximately 1% per year from birth [1].Current medical therapies for heart disease can't replace the injury myocardium tissue with re-new cardiomyocytes.
Stem cell therapies have been investigated as a possible treatment approach for cardiac disease [2][3][4].Although stem cell therapy strategies applied in patients have been extensively investigated in large animal studies [5][6][7][8], clinical success of cardiac cell-therapies remains limited [9].Thus, studies on improving the efficiency of stem cell therapy need to be further investigated.
MicroRNA (miRNA) are a class of 21-25 nucleotides noncoding RNA that bind and inhibit the translation of target messenger RNAs.It is estimated that more than 30% of the human genome may be subjected to be regulated by miRNAs [10].miRNAs are crucial regulators of cellular pathways in differentiation, development, apoptosis, metabolism and proliferation [11][12][13].Growing evidence shows that miRNAs are involved in cardiogenesis and heart pathological process [14][15][16].miR-590 and miR-199a are shown to promote cell cycle re-entry of adult cardiomyocytes ex vivo and enhance the cardiomyocyte proliferation.They stimulated marked cardiac regeneration and almost recovered cardiac functional parameters in a mouse myocardial infarction model [17].MicroRNA also promoted cardiac progenitor cell proliferation and differentiation and facilitated the ESCs to differentiate into cardiomyocyte, which can potentially increase the efficiency stem cell therapy in cardiac diseases.For instance, over-expression of miR-17-92 increases the proliferation in adult cardiac progenitor cells in vivo by two-fold [18].
MiR-155 is located on chromosome 21 and transcribe from the B-cell integration cluster.It can be split into two mature microRNAs: miR-155-3p and miR-155-5p.MiR-155 is the most extensively investigated miRNA in immune cells [19][20][21].Recently, it is also indentified that miR-155 plays a crucial roles in regulation cardiac disease.However the role of miR-155 cardiogenesis is still unknown.
In this study, we explored the expression levels of miR-155-3p during ESC differentiation into cardiomyocyte.The role of miR-155-3p inhibition in cardiogenesis in vitro and its potential mechanism were also investigated.Our novel understanding of the miR-155-3p inhibition in cardiogenesis could partly enlighten a new therapeutic strategy to heart disease.

MEF2C was up-regulated during ESCs differentiation
The expression profiles of MEF2C mRNA and protein during ESCs differentiation were detected using real-time PCR and Western blot.Analysis of MEF2C mRNA expression revealed that MEF2C mRNA level was up-regulated since d6 of cardiac differentiation of ESCs (Figure 1B).When compared to the d0 group, MEF2C mRNA level in d3, d6, d9, d12, and d14 and groups was 1.31±0.36,4.58±0.87,7.29±1.41,and 9.23±1.23,and 8.75±1.37 folds, respectively.There was no significant difference until d6 as compared to the d0 group.MEF2C mRNA keep at high level at d14, but there had no significant difference between d9 and d14.When compared to d6 group, MEF2C mRNA in d9 group was significant higher.Analysis of MEF2C protein expression revealed that MEF2C was significantly up-regulated in d6 group as compared to d0 group.From d6 to d12, It continued to increase and the protein level in d12 group was 5.83±0.88folds when compared to d0 group (Figures 1C and 1D).

Effects of miR-155-3p inhibitor on EBs beating and growth
To evaluate the effect of miR-155-3p inhibition on EBs beating during cardiac differentiation of ESCs, the beating percentage of EBs was counted at d10, d12, and d14.The results indicated that miR-155-3p inhibitor significantly increased percentage of EBs beating (Figure 1E).At d10, the percentage of EBs beating in miR-155-3p inhibitor group (27.09%±2.25%)was significant increased as compared to control group (21.38%±2.03%).With the continuous differentiation, the percentage of EBs beating in both miR-155-3p inhibitor and control groups were increased when compared to the corresponding groups at d10.The percentage of EBs beating in miR-155-3p inhibitor group at d12 and d14 were 53.77%±5.16%(39.25%±4.99% in control) and 68.61%±5.58%(49.79% ±5.70% in control), respectively.Which were significantly increased as compared to the corresponding control groups.These results demonstrated that miR-155-3p inhibitor facilitated the EBs beating.However, as the results showed that miR-155-3p inhibitor did not change the diameters of EBs at d10, d12 and d14 (Figure 1F).

miR-155-3p mimic inhibited the expression of MEF2C
In order to investigate the effects of miR-155-3p mimic on the expression of MEF2C, ESCs were transiently transfected with miR-155-3p mimic.At d3 of EBs (3 days after transient transfection), MEF2C expression was detected using real-time PCR and Western blot.The results showed that miR-155-3p mimic suppressed both MEF2C mRNA (Figure 2A) and protein (Figure 2D) expressions.As compared to the corresponding scramble groups, the MEF2C mRNA and protein levels in miR-155-3p mimic groups were 0.35 ± 0.14 and 0.43±0.20 folds, respectively.These data suggested that miR-155-3p efficiently suppressed the expressions of MEF2C mRNA and protein.

MEF2C was a direct target of miR-155-3p
Sequence alignment of miR-155-3p and its target sites in the 3'-UTR of MEF2C were listed (Figure 5A).To test whether miR-155-3p could directly target the 3'-UTR of MEF2C mRNA in a sequence-specific manner, we generated a luciferase construct harbouring a potential binding site for miR-155-3p and produced three mutant constructs with potential target sites (Figure 5B).Without any mutation, miR-155-3p repressed 58.4% of luciferase activity of the reporter construct.Both mut 1(mutation sites of I and II) and mut 2 (mutation sites of I and III) repressed luciferase activity.However mut 3 (mutation sites of II and III) did not change luciferase activities (Figure 5C).The results suggested that miR-155-3p can repress MEF2C expression through the direct interaction with II or III.
The myocyte enhancer factor 2 (MEF2) proteins belong to the evolutionarily ancient MADS (MCM-1agamous-deficiens-serum response factor) family of transcription factors.MEF2 family is detected in a wide range of tissues, but there are most abundant in striated muscles and brain [22,23].In mammals, this family has four members: MEF2A, MEF2B, MEF2C, MEF2D [24].MEF2C is the first member to be expressed in the somite myotome, appearing initially in mesodermal precursors that give rise to the heart [23].MEF2C regulates the expression of several cardiac structure and contractile proteins which are associated with the cardiogenesis [25].
Murine Mef2c gene is expressed in heart precursor cells before formation of the linear heart tube.In mice homozygous for a null mutation of Mef2c, the heart tube do not undergo looping morphogenesis, the future right ventricle do not form, and a subset of cardiac muscle genes is not expressed.Mef2-null mice exhibit cardiac looping defects and lead to lethality in homozygous mutants by embryonic day 9.5 [26].A dominant-negative fusion protein of MEF2C with the engrailed repression (EnR) domain (MEF2C/EnR) in embryos results in a lack of heart structures and a severe disruption of cardiomyogenesis [27].Similar to the findings in vivo, MEF2C plays crucial role in cardiomyogenesis in vitro.Over-expression of Gli2 up-regulates MEF2C mRNA and protein expression while enhancing cardiac differentiation of P19 ESC [28].HH/GLI2 regulates the expression of Mef2c by recruiting BRG1 to the Mef2c gene, most     probably via chromatin remodeling, to ultimately regulate in vitro cardiomyogenesis [29].Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by over-expression of cardiac transcription factors, including Mef2c, Gata4, and Tbx5 [30].Furthermore, DAPT, a classical notch inhibitor, enhances cardiac reprogramming by increasing binding of the transcription factor MEF2C to the promoter regions of cardiac structural genes [31].Our previous data show that HDAC4 inhibition promotes MEF2C expression in the cardiac differentiation of ESCs [32].In the current study, MEF2C expression was detected during cardiac differentiation of ESCs.The results showed that MEF2C was up-regulated during ESCs differentiation.
Growing evidence indicates that miR-155 is involved in the regulation of cardiac cell.miR-155 can reduce cardiac injury by inhibiting NF-kB pathway during acute viral myocarditis [33].Systemic administration of miR-155 mimic attenuates cardiac dysfunction and improves late sepsis survival by targeting JNK associated inflammatory signaling [34].However, inhibition of miR-155 using antagomiR improved cardiac function and suppressed cardiac apoptosis induced by lipopolysaccharide in mice.MiR-155 is also found to be up-regulated in the plasma of patients with septic cardiac dysfunction.Inhibition of miR-155 represents a novel therapy for septic myocardial dysfunction [35].MicroRNA-155 expression is up-regulated and localized primarily in heart-infiltrating macrophages and CD4(+) T lymphocytes during acute myocarditis.microRNA-155 knockout mice develop attenuated viral myocarditis [36].
MiR-155-3p is down-regulated in heart of fetal and adult cardiac remodeling, an adaptive alteration that results an altered heart structure and function, when compared to control group.By contrast, miR-155-5p is up-regulated [37].It has been shown that miR-155 level in proliferating cardiomyocyte progenitor cells is higher than differentiated cardiomyocyte progenitor [38].However, there was no report regarding to the potential function of miR-155 in cardiac differentiation.In the present study, the expression profile of miR-155-3p during cardiac differentiation of ESCs was investigated.The results indicated that miR-155-3p was down-regulated during ESCs differentiation.Taking these data together, miR-155-3p seems to be involved in the regulation of cardiogenesis.
The target genes of miR-155-3p were investigated by using online bioinformatic tools targetScan (http:// www.targetscan.org/vert_71/).We found that MEF2C was a predicted target gene of miR-155-3p.In this study, we confirmed that miR-155-3p suppressed MEF2C expression using Real-time PCR and Western blot.And luciferase results also showed that MEF2C was a direct target of miR-155-3p.Thus, miR-155-3p regulates cardiogenesis maybe through inhibiting MEF2C.In the present study, we indentified that miR-155-3p was expressed in ESCs and the expression level in ESCs was higher than in cardiocytes differentiated from ESCs.We supposed that miR-155-3p in the ESCs and early cardiac differentiation of ESCs would partly prevent the cardiogenesis through inhibition of MEF2C.Here, we focused on whether miR-155-3p inhibition could be used in vitro to induced cardiomyocyte differentiation.To further unveil the biological function of miR-155-3p inhibition in the process of cardiogenesis, the miR-155-3p inhibitor was stably transfected into ESCs and the cardiac differentiation was explored.The results suggested that miR-155-3p inhibition promoted the cardiomyocyte differentiation and increased MEF2C expression.
RAS proteins are small GTPases that play crucial roles in numerous functions such as control cell proliferation, differentiation and apoptosis.RAS has 3 isoforms, there are HRAS, KRAS, and NRAS.Activation of RAS trigger a broad range of downstream signaling pathways, including the RAF-MEK-Extracellular signalregulate kinase (ERK) pathway [39].In rat ventricular myocytes, activation of ERK1/2 is mediated through the classical Ras/Raf/MEK/ERK pathway, with Ras acting as the upstream trigger [40].
ERK is one of the three MAPKs (ERK, JNK, and p38).It had been identified that ERK was involved in the regulation of cardiac differentiation.Heregulin-beta1 promotes the development of cardiomyocytes derived from ES cell predominantly by activation of MEK-ERK [41].In addition, sustained activation of ERK by 5-Azacytidine contributes to the induction of the differentiation of human mesenchymal stem cell into cardiomyocytes in vitro [42].MAPKs participated in the regulation of MEF2C.MEF2A and MEF2C are preferentially phosphorylatied and activate [43].ERK1/2-RSK2 signaling is a novel mechanism by which neurotrophins activates MEF2C and promotes neuronal survival [44].In myocardial cell line H9C2, inhibition of ERK1/2 decreases alcohol-induced over-expression of GATA4 and MEF2C [45].Our data suggested that ERK1/2 inhibitor decreased the expression of MEF2C protein.Thus, ERK1/2 triggering cardiac differentiation may be related to the regulation of MEF2C.

In vitro differentiation
CGR8 ESCs transfected with miR-155-3p inhibitor or control were dissociated by 0.25% trypsin-0.01%EDTA and suspended in the differentiation medium (ESCs culture medium without LIF).The cell drops (400 cells/20 μl) were placed onto the lids of culture dishes.The lips were reverted and then cover the dishes which contain 2 ml deionized water.After 2 days EB formation by the hanging drop method, Embryoid bodies (EBs) were transferred to non-coated petri dishes for suspension culture up to day 7 (d7).For diameter measurement, EBs were further cultured in non-coated culture dishes.For differentiation assay, EBs were cultured in gelatin-coated 96-well culture plates (1 EB/well).The EBs growth were monitored at day 10 (d10), d12 and d14.The spontaneous rhythmic contraction of EBs was analyzed at d10, d12 and d14.To test the effects of ERK1/2 on the regulation of MEF2C, EBs at d9 were treated with ERK1/2 inhibitor, PD98059 (50 μM) for 24 h.The differentiation experimental protocol was showed in Supplementary Figure 1.

Real-time PCR quantification
Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol.1000 ng total RNA was used as a template for reverse transcription.For protein-coding mRNA, cDNA was obtained with Applied Biosystems Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA).For miR-155-3p, cDNA was obtained with TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA).Real-time quantitative PCR was performed using ABI7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA).The expression levels of miR-155-3p were normalized to U6, while levels of MEF2C, cTnT, nkx2.5, and GATA4 were normalized to GAPDH.The miR-155-3p specific primers and MEF2C, cTnT, nkx2.5, and GATA4 probes were purchased from Applied Biosystems.

Immunofluorescence
The differentiated ESCs (d14) were digested and separated into single cell.After washing with differentiation culture medium for 3 times, the cells were plated into coated coverslips for 12 hours.The coverlips were rinsed with PBS for 3 times and fixed in 4% paraformaldehyde in PBS for 30 minutes and permeabilized with Triton X-100 in PBS for 5 min.The cells were incubated overnight at 4°C with anti-GATA4 (1:200), anti-MEF2C (1:300), anti-cTnT (1:300) or anti-Nkx2.5(1:200) antibody.Subsequently the cells were incubated FITC-or Cy3-conjugated secondary antibody (1:200) for 1 hour at room temperature.The nucleis were stained with 4', 6-diamidino-2-phenylindole (DAPI).The numbers of cTnT, GATA4, MEF2C or Nkx2.5 positive cells on each coverslip were counted in four randomized fields.The cells were observed at 20 times of the microscope.In each field, there were 50-100 cells.The positive cells were identified by the same person.The measurements were done under blind conditions.

Statistical analysis
Data are showed as the mean ± S.D. Comparisons of means between two groups were carried out using a t-test.Statistical comparisons were performed by analysis of variance (ANOVA) with Dunnett's test for multiple comparisons.A value of P < 0.05 was considered to be significant.