Dexamethasone Suppresses Palatal Cell Proliferation through miR-130a-3p

Cleft lip with or without cleft palate (CL/P) is one of the most common congenital birth defects. This study aims to identify novel pathogenic microRNAs associated with cleft palate (CP). Through data analyses of miRNA-sequencing for developing palatal shelves of C57BL/6J mice, we found that miR-449a-3p, miR-449a-5p, miR-449b, miR-449c-3p, and miR-449c-5p were significantly upregulated, and that miR-19a-3p, miR-130a-3p, miR-301a-3p, and miR-486b-5p were significantly downregulated, at embryonic day E14.5 compared to E13.5. Among them, overexpression of the miR-449 family (miR-449a-3p, miR-449a-5p, miR-449b, miR-449c-3p, and miR-449c-5p) and miR-486b-5p resulted in reduced cell proliferation in primary mouse embryonic palatal mesenchymal (MEPM) cells and mouse cranial neural crest cell line O9-1. On the other hand, inhibitors of miR-130a-3p and miR-301a-3p significantly reduced cell proliferation in MEPM and O9-1 cells. Notably, we found that treatment with dexamethasone, a glucocorticoid known to induce CP in mice, suppressed miR-130a-3p expression in both MEPM and O9-1 cells. Moreover, a miR-130a-3p mimic could ameliorate the cell proliferation defect induced by dexamethasone through normalization of Slc24a2 expression. Taken together, our results suggest that miR-130-3p plays a crucial role in dexamethasone-induced CP in mice.


Introduction
Cleft lip with/without cleft palate (CL/P) is a relatively common congenital birth defect in humans that affects approximately 1 in 700 newborns worldwide [1]. The palate is composed of the primary palate, which derives from posterior protrusion of nasal processes, and a pair of secondary palates, derived from the lateral protrusion of the maxillary processes. The development of the secondary palate in mammals includes palatal shelf growth, elevation of the palatal shelves, fusion between paired palatal shelves, disappearance of the medial epithelial seam, and intramembranous ossification of the palatal processes of the premaxilla and palatine bone [2]. Mice have been widely used in the study of palate development, since palate formation and the associated molecular mechanisms of mice are similar to that of humans and occur in a short period of time [3]. In mice, secondary palate development initiates at embryonic day 11.5 (E11.5) with the formation of tissue folds overlying the future palatal shelves within the oral cavity. Cranial neural-crest-derived mesenchymal cells proliferate within the maxillary processes to form the palatal primordium, which further enlarges to develop the palatal shelves. The palatal shelves continuously grow vertically along the sides of the tongue by E13.5 and then, approximately at E14.0, they elevate to a horizontal position above the tongue. At E14.5, the two palatal shelves meet and start to fuse each at the middle of the oral cavity. Finally, the medial epithelial seam disintegrates by either apoptosis, migration toward epithelial triangles at both oral and nasal sides, or epithelial-mesenchymal transition to complete palatal fusion by E16.5 (Supplementary Figure S1). Any failure of these processes leads to CP [3].
The etiology of CL/P is complicated, with both genetic and environmental factors involved as well as their interactions [4,5]. As for environmental factors, maternal exposure to smoking and alcohol consumption are known risk factors for CL/P [3]. In addition, several teratogens (e.g., phenytoin and toxins such as dioxins and heavy metals) are known to cause CP [4]. Environmental factors are thought to influence expression of non-coding RNAs including microRNAs (miRNAs), which are small RNAs with 21-25 nucleotides that regulate the expression of target genes at post-transcriptional level [6]. A number of miRNAs have been found in various species to play roles in a wide array of cellular functions during embryonic development, including palate development [7,8]. We have previously reported that overexpression of miR-374a, miR-133b, and miR-4680-3p inhibits cell proliferation in cultured human palatal mesenchymal cells [9]. In addition, exposure to all-trans retinoic acid (atRA) alters the expression of miR-106-5p [10] and miR-124-3p [11] in mouse embryonic palatal shelves. A growing amount of evidence shows that miRNAs play crucial roles in development and pathological conditions; therefore, it is important to identify how the expression of miRNAs is altered under specific conditions and in presence of chemicals known to cause of CP.
Dexamethasone (DEX) is a synthetic glucocorticoid (GC) clinically used for its antiinflammatory and immunosuppressive actions through interference with various signaling pathways and molecules, including Toll-like receptors and mitogen-activated protein kinases [12]. GC signaling acts as either a transactivator or transrepressor of the target genes under physiological and pathological conditions. GCs in the extracellular fluid diffuse into the cytosol and bind to the GC receptor (GR) in the cytosol. In absence of GCs, nuclear protein GR forms a complex with heat shock protein 70 (HSP70), HSP90, FKBP52, and p23 in the cytosol. In presence of GCs, the GC-GR complex releases HSP70/HSP90/FKB92/p23 and forms a dimer of the GC-GR complex. The activated GC-GR dimer translocates into the nucleus and binds to the glucocorticoid response element (GRE) on the promoter region of its target genes, resulting in the activation of transcription (so called transactivation). In addition, the activated GC-GR complex binds to NFκB (p50/p65) without forming a dimer. This NFκB-conjoined monomeric complex represses transcription by binding to the NFκB response element instead of GRE. Thus, gene expression is suppressed (so called transrepression) [13,14]. Although GCs have tremendous therapeutic usefulness, they are also known for their teratogenicity and toxicity; for example, the oral or systemic administration of corticosteroids increases risk of CL/P two-to nine-fold, a risk of preterm birth or low birth weight [15][16][17], GC-induced osteonecrosis of the femoral head (GIONFH) [18], and GC-induced osteoporosis (GOI) [19]. Furthermore, DEX is known to penetrate the blood-placental barrier and bind to GR in the cytoplasm, causing CP in mice due to suppression of cell proliferation in the palatal mesenchyme [20,21], and craniofacial dysmorphism by upregulated mmp13 expression in zebrafish [22,23]. Although GC treatment induces expression of both genes and miRNAs, the regulatory network of genes and miRNAs remains largely unknown.
Palate formation drastically changes between E13.5 and E14.5, from the cell proliferation phase to the differentiation and extracellular matrix (ECM) secretion phase. During this period, not only morphology but also the gene expression profile is altered according to cellular events. However, it is unclear how gene expression is regulated between E13.5 and E14.5 and whether altered miRNAs are associated with CP. In this study, we first searched for genes (and their functions) regulated by miRNAs during palate development using FaceBase datasets (https://www.facebase.org/, accessed on 27 May 2020). Through the analyses, we identified several miRNAs that were validated using mouse embryonic palatal mesenchymal (MEPM) cells and O9-1 cells, a mouse neural crest cell line. Furthermore, we evaluated whether DEX, atRA, and phenytoin, known teratogens which can induce CP in mice, influenced miRNA expression in MEPM and O9-1 cells.
To test whether overexpression or downregulation of these miRNAs could influence cell growth (crucial at E13.5 for the growth of the palatal shelves), we conducted cell growth assays with a mimic and inhibitor for each miRNA and found that all five miR-449 family miRNAs significantly suppressed cell growth in both MEPM and O9-1 cells ( Figures 1A and S3A). Among them, miR-449c-3p and miR-449c-5p inhibited cell growth more than 30% in both MEPM and O9-1 cells. On the other hand, inhibitors for all of the miR-449 family did not affect cell growth in both MEPM and O9-1 cells ( Figures 1B and S3B). Moreover, we found that overexpression of miR-486b-5p and suppression of miR-130a-3p and miR-301a-3p inhibited cell growth (Figures 1C,D and S3C,D).

DEX Suppresses miR-130a-3p Expression in MEPM and O9-1 Cells
Excessive exposure to certain chemicals such as atRA, phenytoin, and DEX are known to cause CP in mice [4,24,25]. To investigate whether the expression of candidate miRNAs is associated with chemical exposure, we conducted cell growth assays in MEPM and O9-1 cells under treatment with either DEX, atRA, phenytoin, or vehicle. All three chemicals significantly suppressed cell growth in both MEPM and O9-1 cells (Figures 3A,C,E and S5A,C,E). As we expected, miR-130a-3p expression was specifically inhibited under treatment with DEX ( Figures 3B and S5B). The expression of miR-130-3p and miR-301a-3p was upregulated under atRA treatment (Figures 3D and S5D); the expression of miR-486b-5p was significantly suppressed under phenytoin treatment ( Figures 3F and S5F). The expression of miR-449c-5p was not altered and miR-449c-3p was not detected under the test conditions.

Overexpression of miR-130a-3p Restores Cell Proliferation under Treatment with DEX
To evaluate the contribution of miR-130a-3p to cell growth under treatment with DEX, MEPM and O9-1 were treated with a miR-130a-3p mimic. Notably, the miR-130a-3p mimic completely normalized cell growth under treatment with dexamethasone ( Figures 4A,C and S6A,C). The upregulated expression of Slc24a2, but not 1700028K03Rik, was partially restored by treatment with DEX, in MEPM and O9-1 cells (Figures 4B and S6B). Thus, our results indicate that DEX can inhibit cell growth through downregulation of miR-130a-3p in MEPM and O9-1 cells. There are two possibilities for the suppression of cell growth: suppressed cell proliferation or increased cell death. We found that miR-130a-3p contributes to the suppression of cell growth through a decrease of cell proliferation and increase in apoptosis under DEX in both MEPM and O9-1 cells (Figures 4D and S6D).

Slc24a2 Induces Apoptosis in MEPM and O9-1 Cells
The role of Slc24a2 in cell growth has not been evaluated before. Therefore, we tested the effect of overexpression of Slc24a2 in MEPM and O9-1 cells and found that Slc24a2 overexpression inhibited cell growth (Figures 5A and S7A). To clarify the contribution of cell proliferation and apoptosis in cell growth inhibition, we performed BrdU incorporation (for cell proliferation) and TUNEL (for cell death) assays, under Slc24a2 overexpression, in MEPM and O9-1 cells and found that Slc24a2 overexpression induces apoptosis, but does not suppress cell proliferation ( Figures 5C,D and S7C,D). Taken together, our results indicate that DEX inhibits cell growth due to Slc24a2-mediated cell death through downregulation of miR-130a-3p in MEPM and O9-1 cells.
The miRNAs identified in this study have been reported in cancer research. Several studies suggest that miR-130a is important in the progression of several types of cancers and a potential oncogenic miRNA [26]. For example, miR-130a is upregulated in oral squamous cell carcinoma and suppresses expression of TSC1, a tumor suppressor gene, and an miR-301a inhibitor suppresses pancreatic tumor growth in a xenograft model [27]. Overexpression of miR-130a-3p promotes cell proliferation via negative regulation of Runt-related transcription factor 3 (RUNX3) in normal human cervical epithelial cells [28]. Inhibition of miR-130-3p represses cell proliferation by modulating the TGF-β type II receptor in gastric cancer cells [29]. In agreement with these results, miR-301a-3p inhibition suppressed cell proliferation in MEPM and O9-1 cells. miR-486-5p has been detected in various cancer cells [30,31], and its overexpression inhibits cell proliferation in leukemia cells, through targeting forkhead box protein O1 (FOXO1) [32], and accelerates anti-proliferative effects via PIM-1 in breast cancer cells [33]. The miR-449 family was first discovered in the embryonic mouse central nervous system [34]. The binding specificities of miR-449a and miR-449b are very similar, while miR-449c differs from those of others. All three miRNAs regulate the cell cycle and apoptosis. Overexpression of miR-449a induces cell cycle arrest in human bladder cancer cells [35] and suppresses cell proliferation through the regulation of cyclin D1 expression in colon cancers [36]. On the other hand, overexpression of miR-449c inhibits tumorigenesis in non-small cell lung cancer cells [37]. Since these miRNAs are associated with several signaling pathways, these miRNAs may play a crucial role in palate development through the regulation of these signaling pathways.
Prenatal exposure to teratogens such as smoking, alcohol, and chemicals is also known to induce CP in laboratory animals and humans [4,5]. Excessive atRA, DEX, and phenytoin induce CP in mice [24,39,40]. Excessive atRA induces CP through upregulated miR-124-3p expression [11], and DEX induces miR-130b and miR-155 in porcine pre-adipocytes and differentiating 3T3-L1 pre-adipocytes, respectively [41,42]. DEX also inhibits miR-132 expression through TGF-β signaling in pancreatic cancer [43]. Since TGF-β signaling plays crucial roles in palate development [44], a cocktail of miR-132 and miR-130a-3p mimic might be more efficient than a mimic of each miRNA. In addition, the feedback loops between miRNAs and genes and the regulatory networks of miRNAs and genes (e.g., one miRNA regulates expression of multiple genes; gene expression is influenced by multiple miRNAs) may be involved in the rapid regulation of miRNAs by GCs.

BrdU Incorporation and TUNEL Assay
MEPM and O9-1 cells were plated at a density of 15,000/dish (MEPM cells) or 5000/dish (O9-1 cells) and treated with an overexpression vector for mock-[pcDNA3.1 (52535, Addgene, Watertown, MA, USA)] or full-length mouse Slc24a2 (75199, Addgene) under treatment with DEX. After 48 h, the cells were incubated with 100 µg/mL BrdU (B5002, Sigma Aldrich) for 1 h; incorporated BrdU was detected with a rat monoclonal antibody against BrdU (ab6326; Abcam, Cambridge, UK, 1:1000). The Click-iT Plus TUNEL Assay with Alexa 594 (C10618, molecular probes, Thermo Fisher Scientific) was used to detect apoptotic cells, according to the manufacturer's protocol. A total of 12 fields, which were randomly selected from three independent experiments, was used for the quantification of BrdU-positive and TUNEL-positive cells.

Statistical Analysis in Experiments
All experiments were performed independently three times. The statistical significance of the differences between two groups was evaluated using a two-tailed Student t test. Multiple comparisons were evaluated with one-way analysis of variance (ANOVA) adjusted by the post hoc Tukey-Kramer's test. Cell proliferation was analyzed by two-way ANOVA adjusted by the Dunnett's test (for control vs treated group) or Tukey-Kramer's test (for multiple group comparison). A p value less than 0.05 was considered to be statistically significant. Data are represented as mean ± standard deviation in the graphs.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/ijms222212453/s1. Author Contributions: H.Y. contributed to data acquisition, analysis, interpretation, and drafted the manuscript. G.J. contributed to conception, design, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript. A.S. contributed to conception, design, drafted and critically revised the manuscript. J.I. contributed to conception, design, data acquisition and interpretation, draft and critically revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding:
The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The work was supported by the National Institute of Dental and Craniofacial Research (R03DE026208 and R01DE029818 to J.I.).

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Animal Welfare Committee (AWC) and the Institutional Animal Care and Use Committee (IACUC) of UTHealth (AWC 19-0079; 11/01/2019). All mice were maintained at the animal facility of UTHealth.

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.