Involvement of microRNA-4680-3p against phenytoin-induced cell proliferation inhibition in human palate cells

Abstract

Cleft palate (CP) is one of the most common birth defects and is caused by a combination of genetic and/or environmental factors. Environmental factors such as pharmaceutical exposure in pregnant women are known to induce CP. Recently, microRNA (miRNA) was found to be affected by environmental factors. The aim of the present study was to investigate the involvement of miRNA against phenytoin (PHE)-induced inhibition of proliferation in human embryonic palatal mesenchymal (HEPM) cells. We demonstrated that PHE inhibited HEPM cell proliferation in a dose-dependent manner. We found that treatment with PHE downregulated cyclin-D1 and cyclin-E expressions in HEPM cells. Furthermore, PHE increased miR-4680-3p expression and decreased two downstream genes (ERBB2 and JADE1). Importantly, an miR-4680-3p-specific inhibitor restored HEPM cell proliferation and altered expression of ERBB2 and JADE1 in cells treated with PHE. These results suggest that PHE suppresses cell proliferation via modulation of miR-4680-3p expression.

INTRODUCTION

Cleft palate (CP) is one of the most common birth defects worldwide (affects approximately 1:700 live births). The etiology of CP is multifactorial and involves environmental and genetic factors; for example, disruption of signaling pathways including bone morphogenetic protein (BMP), sonic hedgehog protein (SHH), and WNT signaling induces CP (Paiva et al., 2010; Parada and Chai, 2012). A systematic review has revealed that more than 130 genes are associated with CP (Li et al., 2020). Environmental factors such as medication usage, cigarette smoking, and alcohol consumption are well-known risk factors for CP since they lead to suppression of the expression of specific genes or signaling pathways (Dhulipala et al., 2006; Romitti et al., 1999). Although novel gene mutations associated with CP have been reported (Yamaguchi et al., 2021), it remains unclear how environmental and genetic factors affect CP.

MicroRNAs (miRNAs) are small endogenous RNA (18–24 nucleotides long) that regulate post-transcriptional gene expression. Recent studies suggested that miRNAs play crucial roles in palate development in human and mice (Schoen et al., 2017; Suzuki et al., 2019; Li et al., 2020). However, it is still unclear how and which miRNAs play important roles in CP. Alternation of miRNAs were reported to attenuate cell proliferation activity. For instance, Li, Suzuki, and Yoshioka et al. reported that overexpression of hsa-miR-133b, hsa-miR-140-5p, hsa-miR-374a-5p, hsa-miR-381-3p, and hsa-miR-4680-3p inhibits cell proliferation in cultured human embryonic palatal mesenchymal cells (HEPM cells), while mmu-miR-27a-3p, mmu-miR-27b-3p, and mmu-miR-124-3p suppresses cell proliferation in primary mouse embryonic palatal mesenchymal cells (MEPM cells) (Suzuki et al., 2019; Li et al., 2020; Yoshioka et al., 2021b). Moreover, inhibition of mmu-miR-130a-3p and mmu-miR-301a-3p suppresses in MEPM cells (Yoshioka et al., 2021a). These reports suggest that the aforementioned miRNAs may be involved in the pathogenesis of CP.

Phenytoin (PHE) is clinically used as an anti-epilepsy medicine. In addition, PHE was recently used for treatment of gastrointestinal tract fistulas, diabetic foot ulcers, and chronic wounds (Game et al., 2016; Sanad et al., 2019). However, PHE is also known to induce CP (Goldman et al., 1983; Azarbayjani and Danielsson, 2001; Abdollahi Fakhim et al., 2019). Recent studies showed that other medicines such as all-trans retinoic acid (atRA) and dexamethasone alter the expression of miRNAs in HEPM cells and MEPM cells (Yoshioka et al., 2021a, 2021b). Therefore, we hypothesized that PHE may alter the expression of miRNAs. In the present study, we examined the involvement of miRNAs against PHE-induced inhibition of proliferation in HEPM cells.

MATERIALS AND METHODS

Cell culture

HEPM cells were obtained from the JCRB Cell Bank (JCRB9095, Osaka, Japan) and maintained in Minimum Essential Medium Eagle-alpha modification (αMEM; Fujifilm Wako Pure Chemical Corporation (Fujifilm Wako), Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; Biowest, Paris, France) and penicillin/streptomycin (Fujifilm Wako) at 37°C in a humidified atmosphere with 5% CO₂.

Cell proliferation assay

HEPM cells were plated in 96-well plates at a density of 5,000 cells per well. Twenty four hours later, we treated the cells with various concentrations (0.1–100 μM) of PHE (Fujifilm Wako), or vehicle (dimethyl sulfoxide:(DMSO): 0.1%). After 24 hr of treatment, cell viability was evaluated by the Alamar Blue assay (Bio-Rad Laboratories, Hercules, CA, USA).

Apoptosis assay

HEPM cells were plated in 24-well plates at a density of 50,000 cells per well and treated with 100 μM PHE or 0.1% DMSO. After 24 hr of treatment, apoptosis-positive cells were detected using Apotracker Green (BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions and as previously described (Yoshioka et al., 2022). As a positive control, we treated staurosporine for 4 hr. The nuclei were counterstained with Hoechst 33342 (Nacalai Tesque).

Bromodeoxyuridine (BrdU) incorporation assay

HEPM cells were plated in 24-well plates at a density of 50,000 cells per well and treated with 100 μM PHE or 0.1% DMSO. After 24 hr of treatment, the cells were incubated with BrdU (100 μg/mL) for 1.5 hr. Incorporated BrdU was stained with BrdU antibody (Abcam, Cambridge, MA, USA) and fluorescein-conjugated affinipure Goat Anti-Rat IgG (H + L) (Proteintech Japan, Tokyo, Japan). The nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI: Nacalai Tesque). A total of six fields were used for the quantification of BrdU-positive cells.

Quantitative RT-PCR

HEPM cells were plated on 35-mm cell culture dishes at a density of 400,000 cells per plate and treated with 100 μM PHE and vehicle. After 24 hr of treatment, total RNA was extracted using the QIAshredder and miRNeasy Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s protocol (n = 3–5). Reverse transcription-PCR conditions have been previously described (Yoshioka et al., 2023). The levels of the target mRNAs were normalized according to the β-actin levels. The oligonucleotide sequences of the primers used were as follows: human β-actin (NM_001101): sense, 5′-ACCTTCTACAATGAGCTGCGTG-3′, and antisense, 5′-TGGGGTGTTGAAGGTCTCAAAC-3′; human Erb-B2 receptor tyrosine kinase 2 (ERBB2; NM_004448): sense, 5′-ACCTGGATGACAAGGGCTG-3′, and antisense, 5′-CGCTTGATGAGGATCCCAAAG-3′; and human jade family PHD finger1 (JADE1; NM_199320): sense, 5′-CGGGAGCAGGATGTCTTATTTAGG-3′, and antisense, 5′-TGTTCCTGGACTTTGCACACAG-3′. For the analysis of miRNAs, 25 ng of total RNA was reverse-transcribed using miRNA reverse transcription reaction kit (GeneCopoeia, Rockville, MD, USA). miRNA expression was examined using an All-in-One miRNA qRT-PCR Detection Kit (GeneCopoeia) according to the manufacturer’s instructions. Probes for miR-133b (HmiRQP0167), miR-140-5p (HmiRQP0181), miR-374a-5p (HmiRQP0463), miR-381-3p (HmiRQP0479), miR-4680-3p (HmiRQP2296) and U6 (HmiRQP9001) were obtained from GeneCopoeia. The PCR conditions were previously described (Tsukiboshi et al., 2023). The amounts of the target miRNAs were normalized according to the quantity of the miRNA encoding U6.

Western blot analysis

HEPM cells (35 mm dish) were homogenized with 100 μL ice-cold RIPA buffer (Nacalai Tesque) containing a protease inhibitor and centrifuged (18,000 × g for 20 min at 4°C). The resulting supernatants were collected, and protein levels were determined using a BCA protein assay kit (Nacalai Tesque). Protein samples (10 μg) were subjected to 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. anti-Cyclin A (CCNA) (1:1,000 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), anti-mouse Cyclin B (CCNB) (1:1,000 dilution; Santa Cruz Biotechnology), anti-mouse Cyclin D1 (CCND1) (1:1,000 dilution; Santa Cruz Biotechnology), anti-mouse Cyclin E (CCNE) (1:1,000 dilution; Santa Cruz Biotechnology), anti-mouse ERBB2 (1:1,000 dilution; Santa Cruz Biotechnology), anti-rabbit JADE1 (1:2,000 dilution; Proteintech Japan) and anti-mouse β-actin monoclonal antibody (1:2,500 dilution; MBL, Aichi, Japan) were used as primary antibodies for immunoblotting. A peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) and peroxidase-conjugated anti-mouse IgG (Cell Signaling Technology) was used as a secondary antibody (1:10,000 dilution). The immunoreactive bands were visualized by Western Blot Hyper HRP Substrate (Takara Bio, Shiga, Japan). Band intensity was measured using Image J software (NIH, Bethesda, MD, USA).

Immunocytochemistry analysis

HEPM cells were plated on 8-well cell culture slide dishes (Biomedical Sciences Inc., Tokyo, Japan) at a density of 10,000 cells per chamber. After 24 hr later, we treated with PHE or vehicle control for 24 hr. We fixed 4% paraformaldehyde (Nacalai Tesque) for 30 min. Following antigen retrieval using proteinase K (Wako Pure chemical) and blocking of non-specific binding using blocking one histo (Nacalai Tesque), each slide chamber was incubated with mouse anti-ERBB2 monoclonal antibody or rabbit anti-JADE1 (1:200 dilution) as a primary antibody at 4°C overnight. The slice chambers were then incubated with a secondary antibody, anti-mouse IgG-FITC or anti-mouse IgG-FITC (MBL) (1:150 dilution). In addition, sections were counterstained with DAPI for nuclear staining.

Rescue experiments

HEPM cells were plated in 96-well plates at a density of 5,000 cells per well, 35-mm cell culture dishes at a density of 400,000 cells per well, or 8-well cell culture slide dishes at a density of 10,000 cells per chamber. After 3 hr, the cells were transfected with either miR-4680-3p inhibitor (2 pmol) or control miR inhibitor (2 pmol;mirVana, Integrated DNA Technologies, Coralville, IA, USA), using ScreenFect (Fujifilm Wako), according to the manufacturer's protocol. The cells were treated for 24 hr and conducted for further experiments.

Statistical analysis

All statistical analyses were performed using the SPSS software (version 24.0; SPSS, Chicago, IL, USA). Multiple comparisons were evaluated using one-way analysis of variance (ANOVA) followed by the post-hoc Tukey-Kramer test. Statistical significance was set at p < 0.05.

RESULTS AND DISCUSSION

First, to analyze the process of PHE-induced inhibition of cell proliferation in HEPM cells, we conducted cell proliferation assays using various concentrations of PHE. We found that treatment of HEPM cells with PHE inhibited cell viability in a dose-dependent manner (Fig. 1A). PHE is known to inhibit cell proliferation through induction of apoptosis and cell cycle arrest in mice and cell line (Czeizel, 1976; Hu et al., 2011; Khaksary Mahabady et al., 2006; Martinelli et al., 2020). We investigated the involvement of apoptosis and cell cycle by treatment with PHE in HEPM cells. As shown in Fig. 1B, we demonstrated that PHE treatment did not observe apoptosis-positive cells, while treatment with staurosporine activated apoptosis, which suggests that PHE has no effect on apoptosis in HEPM cells. Moreover, cell cycle progression was monitored by BrdU incorporation. We found that BrdU-positive cell was significantly reduced by PHE treatment (Fig. 1C). To further investigate the molecular mechanism of PHE-induced cell cycle arrest, we tested cyclin molecules. Cyclin molecules play a fundamental role in regulating cell cycle events (Koepp et al., 1999). In the cell cycle, CCNA is associated with S phase and mitosis. CCNB is involved with mitosis. CCND1 is associated with G1 phase. CCNE is involved with S phase. We found that PHE had no effect on CCNA and CCNB levels (Fig. 1D). In contrast, we demonstrated that PHE treatment downregulated CCND1 and CCNE (Fig. 1D), thereby indicating that PHE exerted cell cycle arrest through suppression of CCND1 and CCNE in HEPM cells.

Fig. 1

Phenytoin (PHE) suppressed cell proliferation via modulating cell cycle arrest in human embryonic palatal mesenchymal (HEPM) cells. (A) Proliferation of HEPM cells treated with PHE (0–100 μM) for 24 hr. **p < 0.01 and ***p < 0.001 versus control. (B) Apotracker staining (green) of HEPM cells after treatment with 100 μM PHE for 24 hr. The nuclei were counterstained with Hoechst 33342 (blue). Staurosporine was used for positive control. Scale bar, 50 μm. (C) BrdU staining (green) of HEPM cells after treatment with 100 μM PHE for 24 hr. The nuclei were counterstained with Hoechst 33342 (blue). Scale bar, 50 μm. Graph shows the quantification of BrdU-positive cells. **p < 0.01 versus control (n=6). (D) Immunoblotting of HEPM cells after treatment with 100 μM PHE for 24 hr. β-actin served as an internal control. ***p < 0.001 versus control (n=3).

Recently, miRNAs have been reported to be involved in the etiology of CP (Schoen et al., 2017). Suzuki and Li et al. analyzed genes involved in the formation of CP and identified miRNAs associated with the development of CP in humans (Li et al., 2020; Suzuki et al., 2019). They found that the overexpression of miR-133b, miR-140-5p, miR-374a-5p, miR-381-3p, and miR-4680-3p inhibited HEPM-cell proliferation via downregulation of the genes that function downstream of these miRNAs’ target genes in signaling pathways. Yoshioka et al. demonstrated that atRA-induced inhibition of HEPM-cell proliferation occurs through the regulation of miR-4680-3p-ERBB2/JADE1 expression (Yoshioka et al., 2021c). In this study, among the five miRNAs considered, we detected the upregulation of miR-4680-3p expression following treatment with PHE (Fig. 2A). Therefore, the same mechanism may occur by treatment with PHE as well as atRA. To further analyze the involvement of PHE, we measured ERBB2 and JADE1 levels since Yoshioka et al. had reported that atRA attenuated ERBB2 and JADE1 expression by potentiating miR-4680-3p expression (Yoshioka et al., 2021c). ERBB2 is a member of the ERBB receptor tyrosine kinase family, which includes the epidermal growth factor receptor (EGFR) (Yarden and Pines, 2012). The binding of ligands to receptors induces the homo- or heterodimerization of receptors and activates the kinase domain that induces downstream signaling cascades such as MAPK/ERK and PI3K/AKT/mTOR pathways, which are crucial for cell proliferation, migration, and differentiation (Avraham and Yarden, 2011; Arteaga and Engelman, 2014). In addition, in HEPM cells, atRA inhibits cell proliferation by upregulating miR-4680-3p expression, which leads to the suppression of ERBB2 expression and consequent downregulation of the downstream genes functional in the ERK1/2 signaling pathway (Yoshioka et al., 2021c). JADE1 is a transcription factor that has two variants (Panchenko, 2016). The inhibition of JADE1 (both variants) expression by siRNA-knockdown suppressed DNA synthesis in cultured epithelial cell lines and primary fibroblasts (Havasi et al., 2013). Treatment of HEPM cells with atRA downregulates JADE1 expression, leading to decreased cell proliferation via downregulation of CCND1 expression (Yoshioka et al., 2021c). To further investigate the effects of miR-4680-3p on the genes that function downstream of ERBB2 and JADE1, we conducted quantitative RT-PCR analysis (Fig. 2B), immunoblotting analysis (Fig. 2C), and immunocytochemistry analysis (Fig. 2D). We found that treatment with PHE suppressed ERBB2 and JADE1 expression (Fig. 2B). Moreover, the decrease in the levels of ERBB2 and JADE1 was conserved in immunoblotting analysis (Fig. 2C) and immunocytochemistry analysis (Fig. 2D). These results indicate that PHE modulates miR-4680-3p upregulation and ERBB2/JADE1 downregulation.

Fig. 2

Phenytoin (PHE) regulated miR-4680-3p in human embryonic palatal mesenchymal (HEPM) cells. (A) Quantitative RT-PCR for miR-133b, miR-140-5p, miR-374a-5p, miR-381-3p, and miR-4680-3p after treatment of HEPM cells with 100 μM PHE for 24 hr. **p < 0.01. (B) Quantitative RT-PCR for ERBB2, and JADE1 after treatment of HEPM cells with 100 μM PHE for 24 hr. *p < 0.05 and **p < 0.01 versus control. (C) Immunoblotting of HEPM cells after treatment with 100 μM PHE for 24 hr. β-ACTIN served as an internal control. **p < 0.01 versus control (n=3). (D) Immunocytochemistry of HEPM cells after treatment with 100 μM PHE for 24 hr.

To further investigate the contribution of miR-4680-3p, we treated HEPM cells with a miR-4680-3p inhibitor (miR-inhibitor) to examine whether miR- inhibition can restore decreased cell proliferation under PHE treatment conditions. We found that transfection of miR-inhibitor suppressed around 75% in our experimental conditions (Supplementary Fig. S1A). Under this condition, ERBB2 and JADE mRNA expression level was significantly upregulated (Supplementary Fig. S1B). In addition, cell viability was slightly increased (around 5%, Supplementary Fig. S1C). Since ERBB2-overexpressed breast cancer is reported to increase cell proliferation (Gao et al., 2016), ERBB2 upregulated level might associate with HEPM cell proliferation activity. Another possibility is cell line specificity. ERBB2 overexpression might not be involved in cell proliferation under the active condition in HEPM cells. This hypothesis is supported by another group since miR-4680-3p inhibitor did not alter cell viability (Yoshioka et al., 2021c). Further investigation is needed on how ERBB2 regulates HEPM cell viability in the future.

Finally, we treated HEPM cells with a miR-inhibitor with PHE treatment. We demonstrated that a miR-inhibitor partially recovered the reduced cell proliferation by PHE (Fig. 3A). As expected, suppression of ERBB2 and JADE1 was recovered by the miR-inhibitor under PHE treatment conditions (Fig. 3B–3D). We also tested the level of CCND1 and CCNE since PHE downregulated these two proteins (Fig. 1D). MiR-inhibitor normalized CCND1 levels, while we failed to recover CCNE levels (Fig. 3C). Yoshioka et al. previously reported that ERBB2 and JADE1 inhibition using siRNA suppressed cell proliferation of HEPM cells through downregulating CCND1 (Yoshioka et al., 2021c). These results suggest that miR-inhibitor alleviates PHE-induced cell proliferation inhibition by regulating miR-4680-3p-ERBB2/JADE1-CCND1 expression.

Fig. 3

MiRNA inhibitor suppressed phenytoin (PHE)-induced cell proliferation inhibition in human embryonic palatal mesenchymal (HEPM) cells. (A) Proliferation of HEPM cells treated with 100 μM PHE and miR-4680-3p inhibitor for 24 hr. **p < 0.01 and ***p < 0.001. (B) Quantitative RT-PCR for analysis of ERBB2 and JADE1 expression after treatment of HEPM cells with 100 μM PHE and miR-4680-3p inhibitor for 24 hr.*p < 0.05 and **p < 0.01. (C) Immunoblotting of HEPM cells after treatment with 100 μM PHE and miR-4680-3p inhibitor for 24 hr. β-actin served as an internal control. *p < 0.05, **p < 0.01, ***p < 0.001 (n=3). (D) Immunocytochemistry of HEPM cells after treatment with 100 μM PHE and miR-4680-3p inhibitor for 24 hr.

However, the present study also has some limitations: (1) miRNA expression levels: We used other researchers’ datasets for analysis in the present study. Although their datasets helped in the identification of miRNA-gene networks, other miRNAs may have also been affected by treatment with PHE. We need to conduct miRNA-seq to elucidate the mechanism of PHE-induced inhibition of cell proliferation. (2) In vitro experiments: In the present study, we conducted in vitro experiments. In future, in vivo experiments should be performed to contribute the miRNAs by PHE-induced CP. In conclusion, this is the first report to demonstrate the involvement of miR-4680-3p against PHE-induced inhibition of HEPM-cell proliferation. Our results can prove to be instrumental for understanding the etiology of CP.

ACKNOWLEDGMENTS

This research is financially supported by Gifu University of Medical Science Research Grant.

Conflict of interest

The authors declare that there is no conflict of interest.

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