MicroRNA-146a Promotes Embryonic Stem Cell Differentiation towards Vascular Smooth Muscle Cells through Regulation of Kruppel-like Factor 4

Objective Vascular smooth muscle cell (VSMC) differentiation from stem cells is one source of the increasing number of VSMCs that are involved in vascular remodeling-related diseases such as hypertension, atherosclerosis, and restenosis. MicroRNA-146a (miR-146a) has been proven to be involved in cell proliferation, migration, and tumor metabolism. However, little is known about the functional role of miR-146a in VSMC differentiation from embryonic stem cells (ESCs). This study aimed to determine the role of miR-146a in VSMC differentiation from ESCs. Methods Mouse ESCs were differentiated into VSMCs, and the cell extracts were analyzed by Western blotting and RT-qPCR. In addition, luciferase reporter assays using ESCs transfected with miR-146a/mimic and plasmids were performed. Finally, C57BL/6J female mice were injected with mimic or miR-146a-overexpressing ESCs, and immunohistochemistry, Western blotting, and RT-qPCR assays were carried out on tissue samples from these mice. Results miR-146a was significantly upregulated during VSMC differentiation, accompanied with the VSMC-specific marker genes smooth muscle-alpha-actin (SMαA), smooth muscle 22 (SM22), smooth muscle myosin heavy chain (SMMHC), and h1-calponin. Furthermore, overexpression of miR-146a enhanced the differentiation process in vitro and in vivo. Concurrently, the expression of Kruppel-like factor 4 (KLF4), predicted as one of the top targets of miR-146a, was sharply decreased in miR-146a-overexpressing ESCs. Importantly, inhibiting KLF4 expression enhanced the VSMC-specific gene expression induced by miR-146a overexpression in differentiating ESCs. In addition, miR-146a upregulated the mRNA expression levels and transcriptional activity of VSMC differentiation-related transcription factors, including serum response factor (SRF) and myocyte enhancer factor 2c (MEF-2c). Conclusion Our data support that miR-146a promotes ESC-VSMC differentiation through regulating KLF4 and modulating the transcription factor activity of VSMCs. Electronic supplementary material The online version of this article (10.1007/s11596-023-2736-3) contains supplementary material, which is available to authorized users.

MiR-146a has been reported to be involved in inflammatory and immune responses as well as cell proliferation, migration, and apoptosis [17][18][19][20] . It was first reported to negatively regulate the interleukin-1beta-induced inflammatory response in human lung alveolar epithelial cells [21] . In addition, miR-146 has been demonstrated to inhibit nuclear factor-kappa B activity by reducing the metastatic potential of breast cancer cells [22] . Moreover, Yuan et al [23] have revealed that miR-146a modulates the transforming growth factor-beta 1-induced phenotypic differentiation of human dermal fibroblasts. Furthermore, there have been studies investigating the role of miR-146a in other cardiovascular diseases such as myocardial ischemia/reperfusion injury and cardiac hypertrophy/ dysfunction [24,25] . However, little is known about the functional involvement of miR-146a in VSMC differentiation from embryonic stem cells (ESCs). Therefore, this study aimed to determine the role of miR-146a in VSMC differentiation from ESCs.

Western Blotting Analysis
The detailed procedure was similar to that described in our previous studies [13,26] . Briefly, cells were harvested and lysed with RIPA (P0013B, Beyotime, China) supplemented with protease inhibitors (P105539-5g, Aladdin, China). The total protein was quantified using a BCA Protein Assay Kit (P0012S, Beyotime, China). Equal amounts of protein boiled for 10 min (100°C) in 5× SDS loading buffer (P0015, Beyotime, China) were resolved by SDS-PAGE in a 5%-12% tris-glycine gel. The proteins were transferred from the gel to a membrane and blocked with 5% skim milk for 1 h at room temperature. The membrane was incubated with primary antibodies, including antibodies against GAPDH (14C10, CST, USA), smooth muscle alpha-actin (SMαA), 14395-1-AP (Proteintech, USA), smooth muscle myosin heavy chain (SMMHC) (AHP1117, AbD Serotec, UK), smooth muscle 22 (SM22) (Ab14106, Abcam, UK), and h1-calponin (Ab78491, Abcam, UK), respectively. All secondary antibodies were acquired from Proteintech (USA). The GAPDH protein was used as an internal control to ensure equal amounts of protein loading among the groups, and the results were subjected to standard Western blotting analysis.

RT-qPCR for mRNA and miRNA
RT-qPCR was performed as described previously [27] . Briefly, RNA was extracted from the cells using Trizol (12183555, Invitrogen, USA) according to the manufacturer's instructions. Total RNA-and miRNA-specific cDNA syntheses were performed with a PrimeScript RT Master Mix (Perfect Real Time) Kit (RR047A, Takara Bio, Japan) and an miDETECT A Track TM miRNA RT-qPCR Starter Kit (C10712-1, Ribobio, China), separately. The RT-qPCR was run on an ABI Prism 7500 system, using Takara premix Ex Taq Ⅱ (DRR820A, Takara, Japan) or an miDETECT A TrackTM miRNA RT-qPCR Starter Kit. The primers used in this study are listed in table 1. The relative mRNA/microRNA expression level was defined as the ratio to the 18S/U6 expression level, with that of the control sample set as 1.0.

miRNA, siRNA, and Plasmid Transfection
When cultured cells reached 60%-70% confluence, the media were replaced with 1 mL of empty media without antibiotics and maintained for 30 min before transfection. The miRNA, siRNA, or plasmid (BAIAO, China) was transfected into the ESCs using Lipofectamine 3000 (L3000-015, Invitrogen, USA) according to the manufacturer's protocol. The transfected cells were cultured for 48-96 h in DM to allow VSMC differentiation. miR-146a/ mimic and siRNAs (normal control or siRNA-KLF4) were cotransfected using jetPRIME® (Polyplus SA, France).

Luciferase Reporter Assay
Luciferase reporter assays were performed as described in our previous studies [27] . A total of 6×10 4 ESCs were seeded in DM in a collagen-coated 12-well plate. After 96 h, the cells were cotransfected with the luciferase reporter plasmids [serum response factor (SRF) and myocyte-specific enhancer factor-2c (MEF-2c), 0.33 μg/well] and miR-146a, miR-146a inhibitor, or control (100 nmol/L) using Lipofectamine 3000. Renilla (0.1 μg/well, Promega, USA) was included in all transfection assays as an internal control. Luciferase and Renilla activity assays were performed 48 h after transfection. The relative luciferase unit was defined as the ratio of luciferase activity to Renilla activity. The value of the control was set as 1.0.

Animal Study
The detailed procedure for the animal studies was very similar to that described previously [14] . All animal experiments were conducted according to the ARRIVE guidelines. All animal procedures were carried out in accordance with the provisions of the Declaration of Helsinki (as revised in Edinburgh in 2000) and were approved by the Research Ethics Committee of the First Affiliated Hospital of Zhejiang University (Institutional Review Board Approval No. 2013/150). Briefly, C57BL/6J female mice were injected with mimic-or miR-146a-overexpressing ESCs (10 6 cells) mixed with 50 µL of Matrigel. After 10-13 days, the mice were sacrificed and the implanted Matrigel plugs were harvested for SMαA or SMMHC immunofluorescence staining. Partial Matrigel implants were used to extract the total RNA and protein samples for RT-qPCR and Western blotting analysis, respectively.

Statistical Analysis
For all animal experiments, at least 6 animals were used (n≥6); while the cell experiments were performed with at least 3 samples (n≥3). Independent repeated experiments were performed at least in triplicate. All data were checked before analysis to confirm that the data conformed to a normal distribution. The data were expressed as the mean±standard error of mean (SEM) and analyzed using a two-tailed Student's t-test for two-group comparison. Comparisons of different groups were performed using one-way ANOVA followed by Tukey's HSD multiple comparison posthoc test. A value of P<0.05 was considered statistically significant.

miR-146a Promoted VSMC Differentiation In Vivo
To investigate whether miR-146a would promote VSMC differentiation in vivo, Matrigel plugs implanted with ESCs with or without miR-146a overexpression were injected subcutaneously into mice. As expected, more SMαA-positive and SMMHC-positive cells were observed in the implants of miR-146a-overexpressing ESCs ( fig. 2A and 2B), and fewer SMαA-positive and SMMHC-positive cells were presented in the implants of miR-146a inhibitors ( fig. S1A and S1B), as demonstrated by immunofluorescence staining and further ImageJ analysis ( fig. 2C and 2D). In addition, we found that the miR-146a-overexpression implants had a higher expression of smooth muscle differentiation-specific markers, including SMαA, SM22, and SMMHC at the gene level ( fig. 2E) and h1calponin at the protein level ( fig. 2F), further confirming the efficiency of miR-146a overexpression and the importance of miR-146a in VSMC differentiation in vivo. Taken together, these data firmly suggest a regulatory role of miR-146a in VSMC differentiation from ESCs both in vitro and in vivo.

miR-146a Promoted VSMC Differentiation Through Regulation of KLF4
To determine the target gene of miR-146a during   Matrigel plugs implanted with ESCs without (mimic control) or with (miR-146a mimic) miR-146a overexpression were injected subcutaneously into mice. The plugs were harvested at 10th day and were sectioned and subjected to immunofluorescence staining using antibodies against SMαA/SMMHC (blue is DAPI, and red is SMαA/SMMHC). Some immunofluorescence staining data from Matrigel plugs implanted with ESCs without (inhibitor control) or with (miR-146a inhibitor) miR-146a inhibition are in the supplementary materials. A and B: Matrigel plugs with ESCs transfected with miR-146a showed more SMαA/SMMHCpositive cells. C and D: The mean density of SMαA/SMMHC immunofluorescence staining was measured by 3 well-skilled researchers. E and F: Total RNA and protein for RT-qPCR and Western blotting were extracted from Matrigel implants, according to the manufacturer's instructions. The data are represented as the mean±SEM of at least 3 repeats. * P<0.05 vs. control group VSMC differentiation, we tested the expression of potential target genes mentioned in previous studies [28] and according to the TargetScan MicroRNA targeting program (http://www. targetscan.org). We found that KLF4 gene expression was significantly downregulated by miR-146a and displayed a negative correlation with the gene expression levels of miR-146a during VSMC differentiation ( fig. 3A and 3B). These results suggested that KLF4 may be negatively regulated by miR-146a. Based on TargetScan analysis, miR-146a had complementary sequences to the 3′UTR of KLF4's mRNA ( fig. 3C). Our data from the KLF4 inhibition experiment showed that siR-KLF4 significantly downregulated the expression of KLF4 during VSMC differentiation ( fig. 3D), resulting in the upregulation of smooth muscle differentiation-specific markers, including SM22 and SMMHC ( fig. 3E), revealing that knockdown of KLF4 expression may recapitulate the effects of miR-146a during VSMC differentiation. We further investigated whether KLF4 repression is required for miR-146a-mediated VSMC differentiation. Data from the miR-146a mimic and siR-KLF4 cotransfection assay showed that siR-KLF4 exacerbated the promotion of miR-146a expression on VSMC differentiation, which is characterized by the upregulated expression of SM22 and SMMHC (fig. 3F). From these data, we surmised that miR-146a promoted VSMC differentiation through regulation of KLF4.

Regulation of miR-146a-on VSMC Differentiation Possibly through Transcription Factors
SRF and MEF-2c are well-known transcription factors for VSMC gene expression regulation. We tested the transfection efficiency of the miR-146a mimic and the miR-146a inhibitor ( fig. 4A and 4B), and the data showed that the gene expression levels of SRF and MEF-2c were significantly upregulated by the miR-146a mimic (fig. 4C) and downregulated by the miR-146a inhibitor ( fig. 4D), suggesting that miR-146a may play a role in regulating these transcription factors during VSMC differentiation. Additionally, luciferase activity assays with SRF gene reporters also showed that the SRF gene promoter activities were related to the expression of miR-146a ( fig. 4E and 4F), indicating that miR-146a upregulated the transcriptional activity of these genes.

DISCUSSION
VSMCs constitute an important component of blood vessels and carry out the physiological functions of contraction and relaxation of vessels, regulation of blood pressure, and blood flow distribution. Therefore, fully understanding the molecular mechanism of VSMC differentiation is essential in stem cell therapy research for vascular diseases and regenerative medicine. Accumulating evidence has demonstrated that mechanical forces, contractile agonists, extracellular matrix components, and other factors, including reactive oxygen species, Notch family members, and miRs, are responsible for VSMC differentiation. However, the mechanisms that regulate ESC-VSMC differentiation have not been fully elucidated. In the present study, we revealed an important role of miR-146a in regulating VSMC-specific gene expression in vitro and in vivo during VSMC differentiation from ESCs. We provided evidence to support that the identified target gene, KLF4, functions as an important gene repressor during VSMC differentiation from stem cells.
miR-146a has been investigated in different fields, such as inflammatory response, cancer cell migration, and angiogenesis [29][30][31] . We found that miR-146a was upregulated during ESC differentiation towards VSMCs, which was demonstrated by RT-qPCR, in accordance with the rise of miR-34a, miR-29a, and miR-214 levels [13][14][15] . As expected, we confirmed a critical role for miR-146a in VSMC differentiation from ESCs in vitro, using miRNA gain/loss-offunction analyses. Furthermore, by generating miR-146a-overexpression ESC lines and using our wellestablished in-vivo VSMC differentiation model, we provide evidence to support that miR-146a plays an important role in VSMC differentiation in vivo. These data confirm that miR-146a is an important VSMC differentiation regulator.
One of the findings of the current study is that we identified KLF4 as the possible target gene of miR-146a during VSMC differentiation. miR-146a has been reported to inhibit glioma development by targeting Notch1 [32] , which is closely related to vascular biology and differentiation. Other target genes, such as YY1, SP1, and Sirt1, have been reported to be involved in VSMC differentiation by our group [13-15, 33, 34] . Here, we chose these genes as potential targets during VSMC differentiation. According to TargetScan and the RT-qPCR results ( fig. 3A and 3B, fig. S2A and S2B), KLF4 was selected to be the potential target during the ESC differentiation model, which was demonstrated to be a target gene in miR-146a-induced VSMC proliferation [28] . Moreover, our results suggest that KLF4 was negatively regulated by miR-146a and KLF4 siRNA enhanced VSMC marker gene activation induced by miR-146a overexpression, supporting an important role of KLF4 in miR-146a-mediated VSMC differentiation. In addition, we demonstrated that miR-146a regulates the mRNA levels of key transcription factors (SRF and MEF-2c) and promoter activities in VSMCs. Furthermore, several studies have suggested the potential interplay between KLF4 and SRF in the transcriptional regulation of the SMMHC and SM22 genes [35] . Taken together, miR-146a may promote VSMC differentiation from ESCs by attenuating KLF4's negative regulation to VSMC differentiation-specific transcription factors. However, the detailed relationships between KLF4 and these transcription factors were not fully covered in our study.

Open Access
This article is licensed under a Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.