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Using multiple-steps bioinformatic analysis to predict the potential microRNA targets by cardiogenic HoxA11

Wang, Chien-Yinga,b; Liu, Szu-Yuanc,d; Kuo, Fu-Hsuanc,e; Lin, Heng-Fuf; Liu, Chao-Yug; Yang, Yi-Pinga,h,i; Tsai, Fu-Tinga,h; Huang, Wei-Chuna,j,k; Tarng, Yih-Wenl,m; Lin, Hsin-Chin; Lu, Kai-Hsio; Yu, Wen-Chunga,p; Yang, Meng-Yinc,d,q,r,*

Author Information
Journal of the Chinese Medical Association: January 2021 - Volume 84 - Issue 1 - p 68-72
doi: 10.1097/JCMA.0000000000000397
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Abstract

1. INTRODUCTION

Recent studies have shown that pluripotent stem cells and embryonic stem cells are capable of differentiation into functional cardiomyocytes after incubation in cardiac differential medium. There are insufficient numbers of somatic stem cells in adult organs, and the differentiation capacity of somatic stem cells is limiting. Furthermore, the damaged cardiomyocytes or myocardial scars, which are the target areas for myocardial regeneration, may lack the cardiomyogenic differentiation signal that generates functional cardiomyocytes for the clinical application of cell therapy.1,2,3

Rejuvenated state that is capable of avoiding cellular senescence and acquiring pluripotent differentiation potential.4,5 Compared with embryonic stem cells, the ethical issues associated with iPSCs are trivial, and the use of the recipient’s own cells avoids the risk of immune rejection that is generally an obstacle during cell transplantation. Efforts aimed at improving the yield of cardiomyocytes from iPSCs have recently been made by both strictly controlling and standardizing the differentiation protocols to force the differentiation toward specific cardiac subtypes.6,7 Though most of the iPSC-derived cardiomyocytes express myocardial-specific surface markers, the functional reconstruction of these cells, such as regeneration of beating capacity, still requires improvement.

Hox-A11, a repressor of differentiation processes, is expressed in several human organs.8,9 Overexpression of miR133a was shown to differentiate human mesenchymal stem cells into cardiogenic cells.10 Moreover, inhibiting the miRNA maturation pathway by cardiac-specific depletion of Dicer during the early developmental stage in mouse embryos results in immature and dysfunctional hearts and leads to postnatal lethality,11 indicating important roles for miRNAs for normal cardiac function and under pathological conditions.

2. METHODS

2.1. The cell culture for iPSC and cardiomyocyte-lineage differentiation

Murine iPSCs were generated from mouse embryonic fibroblasts derived from 13.5-day-old embryos of C57/B6 mice. For the myocardiogenic induction, 2 × 103 ESCs or iPSCs were hanging drops to form the embryoid body (EB) for 3 days. Then, the EB-like ESCs and/or iPSCs were treated with myocardiogenic induction medium for 2 weeks. The differentiation medium was composed of Iscove’s Modified Dulbecco’s medium (Invitrogen) and was supplemented with 5% fetal calf serum, 0.2 mmol/L l-glutamine, 0.1 mmol/L β-mercaptoethanol, and 0.1 mmol/L nonessential amino acid stock.

2.2. Microarray analysis

Total RNA was extracted from cells using Trizol reagent (Life Technologies, Bethesda, MD, USA) and the Qiagen RNeasy (Qiagen, Valencia, CA, USA) column for purification. Total RNA was reverse-transcribed with Superscript II RNase H-reverse transcriptase (Gibco BRL) to generate Cy3- and Cy5-labeled (Amersham Biosciences Co., Piscataway, NJ, USA) cDNA probes for the control and treated samples, respectively. The labeled probes were hybridized to a cDNA microarray containing 10 000 gene clone immobilized cDNA fragments. Fluorescence intensities of Cy3 and Cy5 targets were measured and scanned separately using a GenePix 4000B Array Scanner (Axon Instruments, Burlingame, CA, USA).

2.3. Multiple-steps bioinformatic analysis

Data analysis was performed using GenePix Pro 3.0.5.56 (Axon Instruments) and GeneSpring GX 7.3.1 software (Agilent, Palo Alto, CA, USA). The average-linkage distance was used to assess the similarity between two groups of gene expression profiles as described below. The difference in distance between two groups of sample expression profiles to a third was assessed by comparing the corresponding average linkage distances (the mean of all pair-wise distances (linkages) between members of the two groups concerned). The error of such a comparison was estimated by combining the standard errors (the standard deviation of pair-wise linkages divided by the square root of the number of linkages) of the average-linkage distances involved. Classical multidimensional scaling was performed using the standard function of the R program to provide a visual impression of how the various sample groups are related.

2.4. Statistical analysis

The results are expressed as mean ± SD. Statistical analyses were performed using the t-test for comparing two groups, and one-way or two-way analysis of variance, followed by Bonferroni’s test, was used to detect differences among three or more groups. The correlation between expression levels and age were analyzed by the Pearson’s correlation coefficient and unpaired Student t test. Results were considered statistically significant at p< 0.05. All analyses were performed using SPSS 12.0.

3. RESULTS

Using multiple-steps bioinformatics analysis to predict programming were subjected to a mRNA profiling of microarray and a literature-based pluripotent stem cell-related gene expression array, as well as predict targeting gene pathway in the cardiogenic development process and cardiomyocyte-lineage differentiation (Fig. 1). We first analyzed the microarray data with bioinformatic measurement (Fig. 1A). Furthermore, the common central pathways and potential candidate genes involved in the cardiomyocyte-lineaged differentiation and development (Fig. 1B). The result of bioinformatics analysis showed the potential candidate genes (Fig. 1C). To determine the downstream target(s) of miR181a, we compared the mRNA array results between the iPSC/control and the iPSC/miR181a and between the iPSC/Spg-control and the iPSC/Spg-miR181a. The genes overexpressed in iPSC/miR181a compared with the iPSC/control and the downregulated genes in the iPSC/Spg-miR181a compared with the iPSC/Spg-control were considered as potential targets of miR181a (Fig. 2). We also predicted several miR181a targets using an online prediction program (www.targetscan.org) and identified Hox-A11 as one such target of miR181a by combining the prediction results with the microarray data (Fig. 2A). Initiation of myoblast differentiation through miR181a directly targeting homeobox protein Hox-A11 has been reported.12 However, the regulatory relationship between miR181a and Hox-A11 in cardiomyocytes has not been previously reported. Our bioinformatics analysis in iPSC-derived cardiomyocytes indicated that Hox-A11 is highly correlated with miR181a and that it potentially mediated the functions of miR181a during the cardiomyocyte differentiation process (Fig. 2B). To further elucidate whether miR-181a directly targets HoxA11 in iPSC-derived cardiomyocytes, we constructed wildtype and mutated forms of Hox-A-11 3′UTR reporter constructs and transfected them into iPSC-derived cardiomyocytes in the presence of miR181a (Fig. 2B). We observed that the wildtype HoxA11 3′UTR-reporter activity was inhibited by miR181a, while mutation of the HoxA11 3′UTR sequence reduced the inhibitory effect of miR181a. Finally, we established an iPSC-derived cardiomyocyte differentiation platform and observed that miR181a is upregulated during the process of cardiomyocyte differentiation. The miR181a approach increased the beating area of cultured iPSC-differentiated cardiomyocytes without affecting the beating frequency (Fig. 2C). Our data demonstrated that the expression levels of the cardiac-specific genes Tbx5, Tbx20, Mef2c, Nkx2.5, cTnt, Cx43 MHC, and MCK increased following miR181a transfection and were decreased by the co-expression of Hox-A11, which suggested a role for miR181a-Hox-A11 signaling in cardiac-specific gene regulation.

F1
Fig. 1:
Using multiple-steps bioinformatic analysis to predict cardiogenic genes with targeting mRNA profiling. A, The microarray data with bioinformatic measurement, including combining with panel module 1 (mouse embryonic stem cells; mESC), Panel module 2 (mouse induced pluripotent stem cells [miPSC]), and panel module 3 (gene list form literature of heart development). B, The common central pathways and potential candidate genes involved in the cardiomyocyte-lineaged differentiation and development. C, The potential candidate genes are including Hox-A11, Cx43, Tbx20, Mef2c, cTnT, Mck, HCN4, and others.
F2
Fig. 2:
Validation of up-stream regulating MicroRNA to target the 3′UTR of HoxA11 mRNA. A, Potential interacted cardiogenic targets of Tbx5, Tbx20, Mal2c, Nkx2.5, cTNT, Cx43, MHC, and MCK in different treatment groups of pluripotent stem cells by using a literature-based comparison of the two microarrays and a software-based gene-lineage system. B, Schematic presentation of a putative miR181a target site in Hox-A11 3′UTR. A literature-based comparison of the two microarrays and a software-based (Targetscan program, www.targetscan.org) comparative analysis of the two datasets. miR181a and its downstream targets Hox-A11 was detected and validated. C, The miR181a is upstream target to bind the 3′UTR of Hoxa11, we used mutated nucleotides at the target site in Hox-A11 3′UTR were labeled in red color (lower panel). Luciferase activity of miR181a-transfected iPSCs treated with wild-type Hox-A11 3′UTR or mutant Hox-A11 3′UTR. *p < 0.05. Data shown here are the mean ± SD of three independent experiments. D, Mir181a is an up-stream regulating microRNA to target the 3′UTR of HoxA11 mRNA during the process of cardiomyocyte differentiation.

4. DISCUSSION

In this present work, our findings have shown that mouse iPSC-differentiated cardiomyocytes, like primary-cultured cells, expressed cardiac markers, including cTnT, Mlc2a, HCN4, and Cx43. Comparing the mouse iPSC-differentiated and primary-cultured cardiomyocytes, we showed that both cardiomyocytes expressed the Cx43 cardiac marker. Cx43 is an integral membrane phosphoprotein and is the main constituent of cardiac gap junctions.13 I/R-associated abnormalities have been linked to Cx43 alterations, such as dephosphorization, degradation, and redistribution away from intercalated disks.14 The blockade of a large portion of Cx43 attenuates ischemic hyper contracture, infarct development, and post-myocardial infarction remodeling.15 In the normal heart, Cx43 is phosphorylated at multiple sites in the carboxy terminus, resulting in its electrophoretic migration at 45 kDa.16 The phosphorylation status of Cx43 regulates its properties, including assembly, trafficking, turnover, and electrical and metabolic coupling.17 Factors and conditions that alter the Cx43 phosphorylation pattern can alter its properties and, by extrapolation, can affect heart function.18 Accumulating reports have revealed the upstream regulators of Cx43 phosphorylation, such as protein kinase C, protein kinase A, protein kinase G, mitogen-activated protein kinases, and miR1, which have been demonstrated to be involved in ischemic and pharmacological post-conditioning signal pathways.15 Using miR-181a, we demonstrated that miR-181a is a novel upstream regulator of both Cx43 protein stability and phosphorylation;19,20 overexpression of miR-181a increased the expression and phosphorylation of Cx43, although the exact mechanism is not clear. The elevated Cx43 protein expression and phosphorylation levels suggest increased cellular repair capacity, as Cx43 has been reported to be involved in protective signaling that prevents cardiomyocyte injury21 and promotes a cardiac injury-resistant state.14 Moreover, we also found that Cx43 mediates miR-181a-dependent upregulation of cell mobility, which is correlated with a previous report showing that Cx43 regulates cell polarity and migration in astrocytes and coronary vascular development.22,23

Hox-A11, a homeobox transcription factor that regulates uterine development, is required for female fertility.12 Recently, Naguibneva et al12 demonstrated that Hox-A11 is a direct target of miR181a during mammalian myoblast differentiation. These investigators demonstrated the upregulation of MyoD, an essential factor for muscle differentiation, through the miR181a-mediated suppression of Hox-A11.8,12 However, the role of miR181a-Hox-a11 signaling in cardiomyocytes is still unclear. Herein, we showed that in iPSC-derived cardiomyocytes, miR181a directly targets the 3′UTR region of Hox-A11. HoxA11 or MicroRNA including miR181a could effectively facilitate the cardiogenic differentiation from pluripotent stem cells.

REFERENCES

1. Ishida M, Miyagawa S, Saito A, Fukushima S, Harada A, Ito E, et al. Transplantation of human-induced pluripotent stem cell-derived cardiomyocytes is superior to somatic stem cell therapy for restoring cardiac function and oxygen consumption in a porcine model of myocardial infarction. Transplantation. 2019; 103:291–8
2. Buikema JW, Lee S, Goodyer WR, Maas RG, Chirikian O, Li G, et al. Wnt activation and reduced cell-cell contact synergistically induce massive expansion of functional human iPSC-derived cardiomyocytes. Cell Stem Cell. 2020; 27:50–63.e5
3. Tang BL. Maturing ipsc-derived cardiomyocytes. Cells. 2020; 9:213
4. Li HY, Chien Y, Chen YJ, Chen SF, Chang YL, Chiang CH, et al. Reprogramming induced pluripotent stem cells in the absence of c-Myc for differentiation into hepatocyte-like cells. Biomaterials. 2011; 32:5994–6005
5. Prigione A, Fauler B, Lurz R, Lehrach H, Adjaye J. The senescence-related mitochondrial/oxidative stress pathway is repressed in human induced pluripotent stem cells. Stem Cells. 2010; 28:721–33
6. Burridge PW, Thompson S, Millrod MA, Weinberg S, Yuan X, Peters A, et al. A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability. PLoS One. 2011; 6:e18293
7. David R, Stieber J, Fischer E, Brunner S, Brenner C, Pfeiler S, et al. Forward programming of pluripotent stem cells towards distinct cardiovascular cell types. Cardiovasc Res. 2009; 84:263–72
8. Yamamoto M, Kuroiwa A. Hoxa-11 and Hoxa-13 are involved in repression of MyoD during limb muscle development. Dev Growth Differ. 2003; 45:485–98
9. Jiang J, Wang X, Gao G, Liu X, Chang H, Xiong R, et al. Silencing of lncRNA HOXA11-AS inhibits cell migration, invasion, proliferation, and promotes apoptosis in human glioma cells via upregulating microRNA-125a: in vitro and in vivo studies. Am J Transl Res. 2019; 11:6382–92
10. Lee SY, Ham O, Cha MJ, Song BW, Choi E, Kim IK, et al. The promotion of cardiogenic differentiation of hMSCs by targeting epidermal growth factor receptor using microRNA-133a. Biomaterials. 2013; 34:92–9
11. Chen JF, Murchison EP, Tang R, Callis TE, Tatsuguchi M, Deng Z, et al. Targeted deletion of Dicer in the heart leads to dilated cardiomyopathy and heart failure. Proc Natl Acad Sci U S A. 2008; 105:2111–6
12. Naguibneva I, Ameyar-Zazoua M, Polesskaya A, Ait-Si-Ali S, Groisman R, Souidi M, et al. The microRNA miR-181 targets the homeobox protein Hox-A11 during mammalian myoblast differentiation. Nat Cell Biol. 2006; 8:278–84
13. Verheule S, van Kempen MJ, te Welscher PH, Kwak BR, Jongsma HJ. Characterization of gap junction channels in adult rabbit atrial and ventricular myocardium. Circ Res. 1997; 80:673–81
14. Srisakuldee W, Jeyaraman MM, Nickel BE, Tanguy S, Jiang ZS, Kardami E. Phosphorylation of connexin-43 at serine 262 promotes a cardiac injury-resistant state. Cardiovasc Res. 2009; 83:672–81
15. Schulz R, Heusch G. Connexin 43 and ischemic preconditioning. Cardiovasc Res. 2004; 62:335–44
16. King TJ, Lampe PD. Temporal regulation of connexin phosphorylation in embryonic and adult tissues. Biochim Biophys Acta. 2005; 1719:24–35
17. Lampe PD, Cooper CD, King TJ, Burt JM. Analysis of Connexin43 phosphorylated at S325, S328 and S330 in normoxic and ischemic heart. J Cell Sci. 2006; 119(Pt 16):3435–42
18. Palatinus JA, Gourdie RG. Diabetes increases cryoinjury size with associated effects on Cx43 gap junction function and phosphorylation in the mouse heart. J Diabetes Res. 2016; 2016:8789617
19. Chien CS, Wang CY, Leu HB, Chien Y, Yang YP, Wang CL, et al. Enhancing induced pluripotent stem cell toward differentiation into functional cardiomyocytes. J Chin Med Assoc. 2020; 83:657–60
20. Mele F, Basso C, Leoni C, Aschenbrenner D, Becattini S, Latorre D, et al. ERK phosphorylation and miR-181a expression modulate activation of human memory TH17 cells. Nat Commun. 2015; 6:6431
21. Ishikawa S, Kuno A, Tanno M, Miki T, Kouzu H, Itoh T, et al. Role of connexin-43 in protective PI3K-Akt-GSK-3β signaling in cardiomyocytes. Am J Physiol Heart Circ Physiol. 2012; 302:H2536–44
22. Homkajorn B, Sims NR, Muyderman H. Connexin 43 regulates astrocytic migration and proliferation in response to injury. Neurosci Lett. 2010; 486:197–201
23. Rhee DY, Zhao XQ, Francis RJ, Huang GY, Mably JD, Lo CW. Connexin 43 regulates epicardial cell polarity and migration in coronary vascular development. Development. 2009; 136:3185–93
Keywords:

Bioinformatic analysis; Cardiogenic HoxA11; Cardiomyocyte; MicroRNAs

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