miR-322 promotes the differentiation of embryonic stem cells into cardiomyocytes

Previous studies have shown that miR-322 regulates the functions of various stem cells. However, the role and mechanism of embryonic stem cell (ESCs) differentiation into cardiomyocytes remains unknown. Celf1 plays a vital role in stem cell differentiation and may be a potential target of miR-322 in ESCs’ differentiation. We studied the function of miR-322An using mESCs transfected with lentivirus-mediated miR-322. RT-PCR results indicated that miR-322 increased NKX-2.5, MLC2V, and α-MHC mRNA expression, signifying that miR-322 might promote the differentiation of ESCs toward cardiomyocytes in vitro. The western blotting and immunofluorescence results confirmed this conclusion. In addition, the knockdown of miR-322 expression inhibited ESCs’ differentiation toward cardiomyocytes in cultured ESCs in vitro. Western blotting results showed that miR-322 suppressed celf1 protein expression. Furthermore, Western blotting, RT-PCR, and immunofluorescence results showed that celf1 may inhibit ESCs’ differentiation toward cardiomyocytes in vitro. Overall, the results indicate that miR-322 might promote ESCs’ differentiation toward cardiomyocytes by regulating celf1 expression.


Introduction
Cardiovascular disease (CVD) is the leading cause of death worldwide. Owing to the lack of regeneration of the human heart, myocardial infarction, and other related conditions can result in the loss of cardiomyocytes, leading to heart failure (Lu et al. 2015;Saleh and Ambrose 2018). Cardiac progenitor cells are pluripotent stem cells present in normal myocardial tissue; they can differentiate into cardiomyocytes, smooth muscle cells, and endothelial cells, which have considerable potential for regenerative restoration (Carvalho et al. 2015;Matsuura et al. 2009). Over the years, stem cell transplantation and differentiation into cardiomyocytes have been promising treatments for myocardial infarction (Cahill et al. 2017;Garbern and Lee 2013;Khodayari et al. 2019). However, although stem cell technology is constantly improving, only a few ESCs can eventually differentiate into cardiomyocytes, which influences the effect of ESCs on myocardial injury treatment (Mummery et al. 2003(Mummery et al. , 2012. Therefore, it is necessary to further study the differentiation mechanisms of ESCs to enhance their therapeutic effects by promoting their differentiation into cardiomyocytes. MicroRNAs (miRNAs) are a class of non-coding small RNA molecules containing approximately 19-25 nucleotides. miRNAs play an important role in the development, metabolism, apoptosis, and differentiation of cells by regulating mRNA (Choi et al. 2017;Wang and Blelloch 2011;Zhang et al. 2019). It has been found that some miRNAs are expressed in ESCs and a variety of adult stem cells (Berardi et al. 2012;Martinez and Gregory 2010). A previous study showed that miRNAs play a pivotal role in the maintenance and orientation of stem cells (Ambros et al. 2003), and can regulate the differentiation of the cardiovascular system (Kwon et al. 2005;Small and Olson 2011).miR-322 has been shown to have beneficial effects in stimulating angiogenesis in tissues damaged by ischemia, such as in the post-myocardial infarction (MI) heart and ischemic hindlimb (Dong et al. 2019;Ghosh et al. 2010). Recent studies have found that Kai Liu and Xiaoping Peng equally contributed to this study. modification of CPCD-derived exosomes by miR-322 protects against myocardial infarction through Nox2-dependent angiogenesis (Youn et al. 2019). In addition, previous studies have shown that miR-322 plays an important role during the early differentiation stage of stem cells (Y. Wang et al. 2019a, b;Zhao et al. 2019).
MiR-322 can increase the expression of Osx and other osteogenic genes by enhancing BMP-2 response, thus playing a vital role in osteogenic differentiation (Gamez et al. 2013). Additionally, it can promote muscle differentiation and inhibit the cell cycle by targeting CDC25A (Sarkar et al. 2010). It represents the highest content of miRNAs in Mesp1-positive progenitor cells and is 25 times higher than that in Mesp1-negative cells. In addition, its expression usually peaks on the fifth day of ESCs' differentiation (Shen et al. 2016). Thus, previous studies have indicated that miR-322 may significantly affect ESCs' differentiation into cardiomyocytes as a regulatory factor. Therefore, it is necessary to study the role of miR-322 in ESCs' differentiation to provide theoretical guidance for the application of ESCs in the treatment of myocardial infarction and other diseases. In this case, the focus is on mESCs using suitable methods to induce their differentiation into cardiomyocytes in vitro.

Culture and differentiation of cell
Mouse embryonic fibroblasts (MEF) were purchased from Shanghai Institutes for Biological Sciences (P0). Mouse embryonic stem cells (mESCs) isolated from C57Bl/6 were obtained from the ATCC (P5). mESC culture and differentiation were performed according to a previously reported protocol (Ivey et al. 2008).

Dual-luciferase reporter gene assay
The celf1-wild type (WT) and celf1-mutated type (Mut) sequences were inserted into the pmiRGLO reporter vector (Synthgene Biotech, Nanjing, China). After co-transfection with the miR-322 mimic or mimic-NC, the luciferase activity of celf1-WT or celf1-Mut was detected using a dualluciferase reporter assay kit (Promega) 48 h after transfection, which was normalized to Renilla luciferase activity.

Real-time PCR
RNA was isolated from the cells using the TransZol Up Plus RNA Kit (TransGen Biotech, Beijing, China). RNA was reverse transcribed using the TransScript® Reverse Transcriptase kit (TransGen Biotech, Beijing, China). mRNA levels were quantified using a 7500 Real-Time PCR instrument (ABI, USA). All primers designed to measure the target gene mRNA levels were purchased from GenScript (Nanjing, China). The primer sequences were as follows: GAPDH ( RT-PCR was performed under the following conditions: denaturation at 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 72 °C for 32 s. Data were analyzed using the comparative threshold cycle (Ct) method. The results are the average of a minimum of three independent experiments performed in triplicate and were normalized using GAPDH.

Western blot
The total cell lysates were used for western blot analysis. To detect proteins, a highly efficient RIPA lysate (Beijing Solarbio Technology, Beijing, China) containing a protease inhibitor cocktail (1:100) was used. Protein concentration was determined using a BCA protein assay kit (Beijing Applygen Technologies Inc., Beijing, China), and the same amount of protein was loaded in western blot procedures, as described below. Western blot analysis was performed using standard protocols with the following primary antibodies: anti-cardiac Troponin T, anti-Nkx2.5, anti-CUG-BP1, and anti-GAPDH (Abcam, Cambridge, UK). The membrane was blocked with 5% skimmed milk in PBST with a primary antibody. After overnight incubation with primary antibodies at 4 °C, the membrane was hybridized with a secondary antibody (1:4000 dilution; Sigma, USA) at room temperature for one hour. Protein signals were detected using a gel imaging system (ImageQuant LAS 4000; USA).

Immunofluorescence
To detect cardiomyocytes derived from ESCs, immunofluorescence staining was performed using primary antibodies against α-actinin (Abcam). Cultured cells were washed with cold PBS (pH 7.4) and fixed with 4% paraformaldehyde (PFA) for 20 min. The cells were then washed with cold PBS containing 0.1% Tween 3 times for 10 min each wash and further blocked with 5/10% serum for 1 h. The cells were then incubated with primary antibodies against α-actinin (Abcam, UK). After washing with PBS, the cells were incubated with secondary antibodies (Beijing Solarbio Technology, Beijing, China), and DAPI (1:5000, Beijing Beyotime Technology, Beijing, China) was used to coverslip the slides. Immunostaining was performed using a fluorescence microscope (Nikon). Quantification of cardiomyocytes derived from ESCs (α-actinin positive) was performed using Photoshop software.

Statistical analysis
All experiments were independently performed at least in triplicate, and the obtained data are reported as the mean ± standard deviation (SD). The results were analyzed by one-way analysis of variance (ANOVA) and Kruskal-Wallis H analysis using SPSS (Statistical Product and Service Solutions) software (IBM, NY, USA). A value of p < 0.05 was considered statistically significant. Figure 1 presents the morphological characteristics of MEF and ESC. EB were planted and observed daily using an inverted microscope. By day 9 of differentiation, the beating area was determined (Fig. 2).

Overexpression of miR-322 promotes the differentiation of ESCs into cardiomyocytes
ESCs were transfected with lentivirus. Using a fluorescence microscope, it was found that photomicrographs of mESC-D3 carrying viral plasmids developed intense green fluorescence (Figs. 3 and 4). RT-PCR analysis revealed that the mRNA expression of OCT-4 and SOX-2 (pluripotent genes) mRNA expression of the miR-322 + (overexpression of miR-322) group showed no significant differences compared to that in the empty and wild groups during differentiation, but NKX-2.5, MLC2V, and α-MHC (cardiac factors) mRNA expression significantly increased (Fig. 5). In addition, western blot analysis indicated that NKX-2.5, an early marker of cardiomyocyte development (Jamali et al. 2001;Lints et al. 1993), was upregulated in the miR-322 + group. The 3′-UTR of celf1 containing the complementary binding sites within miR-322 is shown in Fig. 6A. Notably, the luciferase activity of ESCs that were transfected wild-type (wt) 3′-UTR of celf1 was reduced by miR-322, while the luciferase activity of ESCs that were transfected with mutated-type (mut) 3′-UTR of celf1 was not affected by miR-322 (Fig. 6B). Furthermore, a similar expression alteration of cTnT (cardiac myocyte contractile protein) was observed (Fig. 6C). These results indicate that miR-322 might enhance ESCs' differentiation toward cardiomyocytes.

Down-regulation of miR-322 inhibits the differentiation of ESCs into cardiomyocytes
To further study the role of miR-322 in ESCs' differentiation toward cardiomyocytes, we knocked down miR-322 expression by transfecting lentivirus into mESC-D3 cells. RT-PCR analysis revealed that the OCT-4 and SOX-2 mRNA expression of the miR-322( −) (downregulation of miR-322) group had no significant differences compared with the empty and wild groups during differentiation, but NKX-2.5, MLC2V, and α-MHC mRNA expression were significantly decreased (Fig. 7). In addition, we found that Nkx-2.5 and cTnT protein expression was downregulated in the miR-322( −) group compared to that in the empty and wild groups during differentiation (Fig. 6). These results further indicated that miR-322 might affect ESCs' differentiation into cardiomyocytes.

Overexpression of celf1 inhibits the differentiation of ESCs into cardiomyocytes
MiR-322 and miR-503 are similar in structure and share key targets (Cao et al. 2014;F. Wang et al. 2019a, b). The CELF family is considered to be a target (Cui et al. 2012). Overexpression of miR-503 decreases celf1 protein expression, whereas downregulation of miR-503 increases celf1 protein expression (Cui et al. 2012). Therefore, celf1 is potentially a potential target of miR-322.
MESC-D3 cells were transfected with lentivirus carrying the celf1 gene to examine the role of celf1 in ESCs' differentiation toward cardiomyocytes. RT-PCR results showed that overexpression of celf1 did not affect OCT-4 and SOX-2 mRNA expression in the celf1 + group compared to that in the empty and wild groups during the early differentiation stage (Fig. 8). In addition, NKX-2.5, MLC2V, and α-MHC mRNA expression in the celf1 + group significantly decreased during the differentiation process (Fig. 8). Furthermore, Western blotting results showed that Nkx-2.5 and cTnT were downregulated in the celf1 + group (Fig. 6). These results suggest that celf1 might inhibit cardiac differentiation.

Down-regulation of celf1 promotes the differentiation of ESCs into cardiomyocytes
To further study the role of celf1 in ESCs' differentiation, we inhibited celf1 expression. RT-PCR results showed that OCT-4 and SOX-2 mRNA expression in the celf1( −) (downregulation of celf1) group were not significantly different from those in the empty and wild groups during differentiation, but NKX-2.5, MLC2V, and α-MHC mRNA expression were significantly increased (Fig. 9). In addition, we found that NKX-2.5 and cTnT protein expression was upregulated in the celf1( −) group compared to that in the empty and wild groups during differentiation (Fig. 6). These results further indicate that celf1 might regulate the maturation of cardiomyocytes.

miR-322 regulates celf1 expression in the differentiation of ESCs into cardiomyocytes
The relationship between miR-322 and celf1 remains unclear. Therefore, ESCs were prepared to express miR-322 and celf1. Western blotting results showed that overexpression of miR-322 by lentivirus suppressed celf1 protein expression compared to that in the empty and wild groups. In addition, celf1 protein expression was upregulated in the miR-322( −) group (Fig. 10). These results suggest that miR-322 promotes ESCs' differentiation into cardiomyocytes by inhibiting celf1 expression. Fig. 2 A, B, C, D, E, and F are, respectively, 12 h, 1 day, 3 days, 5 days, 7 days, and 9 days after the EB seed plates are planted. : myocardial cell beating area

miR-322 and celf1 regulate the differentiation of ESCs into cardiomyocytes
To further examine the role of miR-322 and celf1 in ESCs' differentiation into cardiomyocytes, we performed immunofluorescence staining. The cardiac structure gene α-actinin was detected on the tenth day of differentiation and used to reflect the physiological status of cardiomyocytes. Immunofluorescence results revealed that both miR-322 and celf1 increased the α-actinin protein expression. These results indicate that miR-322 and celf1 could regulate the differentiation of ESCs into cardiomyocytes (Fig. 11).

Discussion
ESCs provide a vital and renewable cell source capable of repairing or replacing damaged tissues from degenerative diseases, such as Alzheimer's disease, stroke, and MI, which can potentially revolutionize medicine (Duncan and Valenzuela 2017;Hao et al. 2014). Myocardial infarction can result in myocardial necrosis and poor prognosis. ESCs' transplantation after myocardial infarction can promote cardiac repair and enhance cardiac function by maintaining ESCs' survival and migration, enhancing their differentiation toward cardiomyocytes and inducing angiogenesis (Pal 2009;Zhang et al. 2018). Thus, it is necessary to further study the mechanisms of ESCs' differentiation. This study investigated the effects of miR-322 and celf1 on the differentiation of ESCs into cardiomyocytes.
Heart development involves a succession of cellular migration, fusion, and specific differentiation. Several key regulatory factors such as NKX2.5, Tbx5, and α-MHC play vital roles in cardiac development (Fujikura et al. 2002;Zhao et al. 2008). ESCs can generate several fully differentiated cells in the body, including cardiomyocytes, which can decrease ischemic myocardium and improve heart function Ng et al. 2010). Various studies have concluded that miRNAs regulate ESCs' differentiation by controlling a large number of genes via posttranscriptional regulation (Cordes et al. 2009;Yang et al. 2011). Previous studies have indicated that miR-322 plays a protective role in MI and regulates ESCs' differentiation (Dong et al. 2019;Youn et al. 2019). Meanwhile, this study CELF, also known as CUGBP, is an RNA-binding protein. Members of this protein family regulate pre-mRNA alternative splicing and may also be involved in mRNA editing and translation (Barreau et al. 2006;Good et al.  2000; Ladd et al. 2001). As a member of the CELF family, celf1 can regulate embryonic development, cardiomyocyte maturation, adipose tissue, and skeletal muscle differentiation (Blech-Hermoni et al. 2016;Marquis et al. 2006). Celf1 abnormalities are involved in the pathogenesis of various diseases (Blech-Hermoni et al. 2013;Dasgupta and Ladd 2012). Previous studies have shown that celf1 can mediate connexin 43 mRNA degradation in dilated cardiomyopathy (Chang et al. 2017). In addition, celf1 contributes to dilated cardiomyopathy by regulating gap junction integrity (Jeffrey and Sucharov 2018). Furthermore, putative miR-322/-503 target sites on the Fig. 6 The protein expression of Nkx-2.5 and cTnT in ESCs with overexpression or knockdown of miR-322 or celf1 at 10 days after virus transfection. A has-miR-322 and its putative binding sequence in the 3′ UTR of celf1; B Luciferase assay of ESCs co-transfected with miR-322 mimics and the luciferase reporter; C The expression of Nkx-2.5 and cTnT in ESCs   Fig. 7 The mRNA expression of OCT-4, SOX-2, NKX-2.5, MLC2V, and α-MHC during the differentiation of ESCs toward cardiomyocytes in miR-322( −), wild and empty group. The values in each graph are represented as mean ± SD. n ≥ 3, *P < 0.05 vs empty group  Celf1 3′-UTR were predicted using several computational programs, such as Miranda and TargetScan (Sarkar et al. 2010). Experiments were performed in vitro to investigate the role of celf1 in ESCs' differentiation toward cardiomyocytes. They found that overexpression of celf1 had little effect on pluripotency (Oct4 and Sox2) but significantly increased the mRNA expression of Nkx2-5, MLC2V, and α-MHC. Moreover, celf1 inhibits ESCs' differentiation toward cardiomyocytes. These results further indicated that miR-322 could inhibit celf1 protein expression, signifying that celf1 could be a target of miR-322.
This study showed that miR-322 might promote the differentiation of ESCs into cardiomyocytes by regulating celf1. Furthermore, the results suggest that overexpression of miR-322 might enhance the protective role of ESCs' transplantation after myocardial infarction. Thus, this study provides theoretical guidance for the clinical application of ESCs in myocardial infarction treatment. Nevertheless, further experiments need to be designed to support this study.
Author contribution KL and XP contributed equally to this study. KL conceived the study and designed the experiments. LL and XP completed the experiments, analyzed the data, and wrote the manuscript. KL analyzed the data. KL and XP discussed the results and revised the manuscript accordingly.
Funding This study was supported by the National Natural Science Foundation of China (No. 81460047) and Regional Science Foundation (No. 81660359).

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations
Ethics approval and consent participate Not applicable.

Animal ethics
The study was performed in accordance with the ethical standards laid down in the Declaration of Helsinki and approved by the ethics committee of the Ganzhou People's Hospital.

Conflict of interest
The authors declare no competing interests.