Genes Related to Frontonasal Malformations Are Regulated by miR-338-5p, miR-653-5p, and miR-374-5p in O9-1 Cells

Abstract

Frontonasal malformations are caused by a failure in the growth of the frontonasal prominence during development. Although genetic studies have identified genes that are crucial for frontonasal development, it remains largely unknown how these genes are regulated during this process. Here, we show that microRNAs, which are short non-coding RNAs capable of targeting their target mRNAs for degradation or silencing their expression, play a crucial role in the regulation of genes related to frontonasal development in mice. Using the Mouse Genome Informatics (MGI) database, we curated a total of 25 mouse genes related to frontonasal malformations, including frontonasal hypoplasia, frontonasal dysplasia, and hypotelorism. MicroRNAs regulating the expression of these genes were predicted through bioinformatic analysis. We then experimentally evaluated the top three candidate miRNAs (miR-338-5p, miR-653-5p, and miR-374c-5p) for their effect on cell proliferation and target gene regulation in O9-1 cells, a neural crest cell line. Overexpression of these miRNAs significantly inhibited cell proliferation, and the genes related to frontonasal malformations (Alx1, Lrp2, and Sirt1 for miR-338-5p; Alx1, Cdc42, Sirt1, and Zic2 for miR-374c-5p; and Fgfr2, Pgap1, Rdh10, Sirt1, and Zic2 for miR-653-5p) were directly regulated by these miRNAs in a dose-dependent manner. Taken together, our results highlight miR-338-5p, miR-653-5p, and miR-374c-5p as pathogenic miRNAs related to the development of frontonasal malformations.

1. Introduction

Both genetic and environmental factors contribute to the etiology of frontonasal anomalies. Although whole-genome sequencing is commonly applied in medical research, the causes of nearly 70% of all birth defects in humans, including frontonasal anomalies, remain unknown. In addition, more than half of all birth defects involve craniofacial deformities, and the range of variation for any given facial trait often displays a substantial overlap between affected and healthy individuals [1].

The frontonasal prominence, which gives rise to the mid- and upper face, starts to develop with the thickening of the surface ectoderm in the frontonasal process at the 5th week of gestation in humans, and at embryonic day 10 (E10.0) in mice, and further develops into two bilateral nasal placodes, which form the lateral and medial nasal processes that are separated by the nasal pits at the center [2,3,4]. Frontonasal malformations including frontonasal hypoplasia, frontonasal dysplasia, and hypotelorism are caused by hypoplastic growth of the frontonasal prominence, and consequently of the nasal placodes [1,2]. Cranial neural crest (CNC) cells, which constitute the majority of the mesenchymal cells present in the craniofacial region that can give rise to various cell types [5], play a crucial role in the formation and growth of the frontonasal process [6]. To date, various genetic factors contributing to frontonasal malformations have been identified through mouse genetic studies; however, it remains elusive how epigenetic factors adversely influence gene expression during frontonasal development.

The development of the frontonasal region is regulated by a gene network, and a failure in this process results in frontonasal anomalies with varying severities. Although mild cases of frontonasal anomalies are harmless variations, in severe cases the condition is very impactful on both appearance and health, with effects such as obstructive sleep apnea, malocclusion, and dry eyes. Human and mouse genetic studies suggest that various genes are involved in frontonasal development [2]; however, the regulatory mechanisms of genes associated with frontonasal development remain largely unknown. Mice with loss of all mature microRNAs, which are short non-coding RNAs that regulate gene expression at the post-transcriptional level and fine-tune the expression of ~30% of all mammalian protein-encoding genes [7,8,9], in CNC cells (Dicer^F/F;Wnt1-Cre mice) display severe frontonasal dysplasia [10,11,12], indicating that miRNAs play crucial roles in frontonasal development. Recent studies show that miRNAs are indeed essential for the survival of CNC cells during craniofacial development, and disruption of miRNA function in these cells results in frontonasal deformities in mice [3,13]; however, it is still largely unknown which and how miRNAs contribute to frontonasal development.

In this study, we aimed to identify miRNAs, and test their functional significance in the regulation of their downstream genes, that contribute to the pathogenesis of frontonasal malformations.

2. Materials and Methods

2.1. Gene Search

To identify a set of genes related to frontonasal malformations in mice, we searched the Mouse Genomic Informatics (MGI) database (https://www.informatics.jax.org, 1 February 2024) using ‘frontonasal hypoplasia’, ‘frontonasal dysplasia’, and ‘hypotelorism’ as search terms. All genes were further evaluated from the referenced literature for validation purposes.

2.2. Bioinformatic Analysis

miRNA target gene regulatory mechanisms were predicted using miRTarbase, miRanda, PITA, and TargetScan, with the Fisher’s exact test for determining the significance level of the shared genes between miRNA targets and genes related to mouse frontonasal malformations, as previously described [14]. The Benjamini–Hochberg procedure was used for multiple test correction. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (http://www.genome.jp/kegg, 1 February 2024) was conducted to identify shared biological system(s) with ShinyGO ver0.80 [15,16,17]. A Gene Ontology (GO) enrichment analysis (http://www.geneontology.org, 1 February 2024) was conducted using the ShinyGO ver0.80 [15] for cell component (CC), biological process (BP), and molecular function (MF). Significantly enriched categories for the genes were filtered with a false discovery rate (FDR)-adjusted p-value < 0.05 using the hypergeometric test and at least four genes related to frontonasal malformations. Hierarchical level 4 was used at the cut-off in order to avoid too general GO terms.

2.3. Cell Culture

O9-1 cells, a neural crest cell line (SCC049, Millipore Sigma, Burlington, MA, USA), were maintained in medium for embryonic stem cells (ES-101-B, Millipore Sigma) at 37 °C in a humidified atmosphere with 5% CO₂, as previously described [18].

2.4. Cell Proliferation Assay

O9-1 cells were treated with a mimic for the negative control (4464061; mirVana miRNA mimic, ThermoFisher Scientific, Waltham, MA, USA), miR-338-5p, miR-653-5p, or miR-374c-5p (4464066; mirVana miRNA mimic), or an inhibitor for the negative control (4464079; mirVana miRNA mimic), miR-338-5p, miR-653-5p, or miR-374c-5p (4464084; mirVana miRNA inhibitor), using the Lipofectamine RNAiMAX transfection reagent (ThermoFisher Scientific) according to the manufacturer’s protocol. Cell proliferation was measured using the Cell Counting Kit 8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) 24, 48, or 72 h after each treatment (n = 6 per group), as previously described [18].

2.5. Bromodeoxyuridine (BrdU) Incorporation Assay

O9-1 cells were plated onto 35 mm dishes at a density of 10,000/dish and treated with a mimic for a negative control (4464061; mirVana miRNA mimic, ThermoFisher Scientific), miR-338-5p, miR-374c-5p, or miR-653-5p (4464066; mirVana miRNA mimic), using the Lipofectamine RNAiMAX transfection reagent (ThermoFisher Scientific) according to the manufacturer’s protocol. After 72 h, the cells were incubated with BrdU for 1 h. Incorporated BrdU was stained with a rat monoclonal antibody against BrdU (ab6326; Abcam, 1:1000), as previously described [19]. A total of ten fields, which were randomly selected from three independent experiments, were used for the quantification of BrdU-positive cells. Hematoxylin was used for counter staining. Color images were taken with a light microscope (BX43, Olympus, Tokyo, Japan).

2.6. Immunocytochemical Analysis

O9-1 cells were plated onto 35 mm glass-bottom dishes at a density of 10,000/dish and treated with a mimic for a negative control (4464061; mirVana miRNA mimic, ThermoFisher Scientific), miR-338-5p, miR-374c-5p, or miR-653-5p (4464066; mirVana miRNA mimic), using the Lipofectamine RNAiMAX transfection reagent (ThermoFisher Scientific) according to the manufacturer’s protocol. The immunocytochemical analysis was performed as previously described [19], using rabbit monoclonal antibodies against Ki-67 (ab16667, Abcam, 1:600). Hematoxylin was used for counter staining. Color images were taken under a light microscope (BX43, Olympus).

2.7. Terminal 2′-Deoxyuridine, 5′-Triphosphate (dUTP) Nick-End Labeling (TUNEL) Staining

O9-1 cells were plated onto 35 mm dishes at a density of 10,000/dish and treated with a mimic for a negative control (4464061; mirVana miRNA mimic, ThermoFisher Scientific), miR-338-5p, miR-374c-5p, or miR-653-5p (4464066; mirVana miRNA mimic), using the Lipofectamine RNAiMAX transfection reagent (ThermoFisher Scientific) according to the manufacturer’s protocol. The Click-iT Plus TUNEL Assay with Alexa 594 (C10618, Molecular Probes) was used to detect apoptotic cells, as previously described [20]. A total of four fields, which were randomly selected from two independent experiments, were used for the quantification of TUNEL-positive cells. Images were taken with a confocal microscope (Ti-E, Nikon, Tokyo, Japan).

2.8. Quantitative RT-PCR

O9-1 cells were treated with either mimic or inhibitor for miR-338-5p, miR-653-5p, miR-374c-5p, or negative control at 80% confluence, as previously described [18]. Twenty-four hours after the transfection, total RNA was extracted with the QIAshredder and miRNeasy Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s protocol (n = 6 per group). Extracted total RNAs were converted to cDNA, and gene expression was analyzed with quantitative RT-PCR (qRT-PCR) using the CFX96 Touch Real-Time PCR Detection system (BioRad). The PCR primers used in this study are listed in Supplementary Table S1. The expression of each gene was normalized with Gapdh expression. The miRNA expression was measured with TaqMan Fast Advanced Master Mix and TaqMan Advanced miRNA cDNA Synthesis Kit (ThermoFisher Scientific) or All-in-One miRNA qRT-PCR Reagents Kits (GeneCopoeia, Rockville, MD, USA), according to the manufacturer’s protocol.

2.9. Taqmann Assay

miRNA expression was measured in the frontonasal primordium of C57BL/6J mice at E10.5, E11.5 and E12.5, using the Taqman Fast Advanced Master Mix and Taqman Advanced miR cDNA Synthesis Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. Probes for miR-224-3p (mmu481009_mir), miR-383-3p (mmu481150_mir), miR-6951-3p (mmu482850_mir), and miR-7116-3p (466435_mat) were obtained from Thermo Fisher Scientific. The expression was normalized with U6 (4427975).

2.10. Statistical Analysis

Statistical analysis between two groups was performed with a two-tailed non-parametric Student’s t-test. Multiple comparisons were conducted with one-way analysis of variance (ANOVA) with the Tukey–Kramer post hoc test. Cell proliferation assays were analyzed with a two-way ANOVA. All results were obtained from three independent experiments; all experimental data were analyzed using the Prism software (GraphPad Software, Prism 10.1.2). A p-value < 0.05 was considered statistically significant. For all graphs, the data are represented as mean ± standard deviation (SD) with n = 6 per group.

3. Results

3.1. Identification of a Set of Genes Related to Frontonasal Malformations

To collect information on mouse genes related to frontonasal malformations, we conducted a search of the MGI database using the terms ‘frontonasal hypoplasia’, ‘frontonasal dysplasia’, and ‘hypotelorism’, and then confirmed this information with the referenced literature. As a result, a total of 25 genes related to frontonasal malformations (4 genes in frontonasal hypoplasia, 6 genes in frontonasal dysplasia, and 15 genes in hypotelorism) were identified (Figure 1A and

Table 1. Genes related to frontonasal malformations.

Gene Symbol	References (PMID)	Chromosome	Description
Frontonasal hypoplasia (4 genes)
Bmp4	24785830	14	bone morphogenetic protein 4
Cdc42	28326341	4	cell division cycle 42
Ndst1	16020517	18	N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 1
Rdh10	17473173	1	retinol dehydrogenase 10 (all-trans)
Frontonasal dysplasia (6 genes)
Alx1	35127681	10	ALX homeobox 1
Alx3	19409524	3	aristaless-like homeobox 3
Alx4	25673119	2	aristaless-like homeobox 4
Fgfr2	11274405	7	fibroblast growth factor receptor 2
Ndst3	18385133	3	N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 3
Tcof1	16938878	18	treacle ribosome biogenesis factor 1
Hypotelorism (15 genes)
Boc	21183473	16	biregional cell adhesion molecule-related/downregulated by oncogenes (Cdon) binding protein
Cdon	21183473	9	cell adhesion molecule-related/downregulated by oncogenes
Disp1	15269168	1	dispatched RND transporter family member 1
Ift27	25446516	15	intraflagellar transport 27
Ift57	17027958	16	intraflagellar transport 57
Lrp2	26107939	2	low-density lipoprotein receptor-related protein 2
Nosip	25546391	7	nitric oxide synthase-interacting protein
Pgap1	10529425	1	post-GPI attachment to proteins 1
Shh and Sulf1 and Sulf2	18213582	5	sonic hedgehog
		1	sulfatase 1
		2	sulfatase 2
Shh and Six3	18694563	17	sine oculis-related homeobox 3
Sirt1	28273169	10	sirtuin 1
Wdr11	29263200	7	WD repeat domain 11
Zic2	29992973	14	zinc finger protein of the cerebellum 2

PMID: PubMed identifier.

). An MGI mouse phenotype (MP) analysis showed that these genetic mutations were also often involved in ocular hypotelorism (MP:0006197), abnormal medial nasal prominence morphology (MP:0009903), and absent nasal septum (MP:0004872) (Figure 1B and

Table 2. Enrichment analysis for genes related to frontonasal malformations.

Enrichment FDR	Gene Number	Pathway Genes	Pathway	Genes
MGI enrichment
1.70 × 10−15	7	13	MP:0006197 ocular hypotelorism	Ift27, Sirt1, Disp1, Ift57, Wdr11, Zic2, Pgap1
1.01 × 10−8	4	8	MP:0009903 abnormal medial nasal prominence morphology	Tcof1, Rdh10, Wdr11, Pgap1
2.83 × 10−6	3	7	MP:0004872 absent nasal septum	Tcof1, Rdh10, Lrp2
3.62 × 10−6	3	8	MP:0011759 absent Rathke’s pouch	Bmp4, Fgfr2, Pgap1
3.62 × 10−6	3	8	MP:0006290 proboscis	Ndst1, Zic2, Pgap1
3.84 × 10−9	5	20	MP:0005163 cyclopia	Shh, Disp1, Wdr11, Zic2, Pgap1
1.50 × 10−6	4	27	MP:0004592 small mandible	Ift27, Tcof1, Wdr11, Pgap1
1.01 × 10−12	8	60	MP:0005157 holoprosencephaly	Shh, Lrp2, Disp1, Cdon, Six3, Wdr11, Zic2, Pgap1
1.64 × 10−7	5	47	MP:0000107 abnormal frontal bone morphology	Shh, Tcof1, Disp1, Fgfr2, Alx4
5.49 × 10−7	5	61	MP:0003052 omphalocele	Ift27, Bmp4, Lrp2, Alx4, Ndst1
8.23 × 10−7	5	67	MP:0003743 abnormal facial morphology	Disp1, Fgfr2, Wdr11, Ndst1, Pgap1
9.08 × 10−12	9	141	MP:0000783 abnormal forebrain morphology	Shh, Tcof1, Rdh10, Lrp2, Disp1, Alx1, Cdon, Wdr11, Pgap1
4.79 × 10−9	7	110	MP:0003232 abnormal forebrain development	Bmp4, Tcof1, Lrp2, Six3, Ndst1, Zic2, Pgap1
2.87 × 10−6	5	89	MP:0000433 microcephaly	Shh, Tcof1, Wdr11, Zic2, Pgap1
3.62 × 10−6	5	95	MP:0000074 abnormal neurocranium morphology	Shh, Tcof1, Disp1, Fgfr2, Alx4
1.08 × 10−10	9	191	MP:0001293 anophthalmia	Shh, Bmp4, Tcof1, Lrp2, Six3, Wdr11, Ndst1, Zic2, Pgap1
4.79 × 10−9	8	196	MP:0000428 abnormal craniofacial morphology	Shh, Sirt1, Tcof1, Rdh10, Fgfr2, Cdon, Six3, Wdr11
1.64 × 10−7	7	200	MP:0000438 abnormal cranium morphology	Shh, Cdc42, Tcof1, Fgfr2, Six3, Ndst1, Pgap1
2.05 × 10−8	8	246	MP:0002092 abnormal eye morphology	Shh, Sirt1, Bmp4, Tcof1, Rdh10, Lrp2, Ndst1, Pgap1
6.25 × 10−9	9	324	MP:0011090 perinatal lethality, incomplete penetrance	Shh, Ift27, Sirt1, Bmp4, Lrp2, Cdon, Alx4, Wdr11, Pgap1
KEGG enrichment
2.80 × 10−7	5	58	Hedgehog signaling pathway	Shh, Boc, Lrp2, Disp1, Cdon
8.51 × 10−3	2	24	Glycosaminoglycan biosynthesis	Ndst3, Ndst1
2.93 × 10−2	2	63	Basal cell carcinoma	Shh, Bmp4
1.87 × 10−2	3	181	Axon guidance	Shh, Cdc42, Boc
3.11 × 10−2	4	542	Pathways in cancer	Shh, Cdc42, Bmp4, Fgfr2
GO enrichment (BP)
1.64 × 10−13	10	132	Embryonic skeletal system development	Shh, Sulf2, Alx3, Sulf1, Bmp4, Rdh10, Fgfr2, Alx1, Alx4, Ndst1
2.84 × 10−9	7	98	Embryonic skeletal system morphogenesis	Alx3, Bmp4, Rdh10, Fgfr2, Alx1, Alx4, Ndst1
4.78 × 10−8	7	156	Appendage morphogenesis	Shh, Alx3, Sulf1, Bmp4, Rdh10, Alx1, Alx4
4.78 × 10−8	7	156	Limb morphogenesis	Shh, Alx3, Sulf1, Bmp4, Rdh10, Alx1, Alx4
6.96 × 10−8	7	166	Morphogenesis of embryonic epithelium	Shh, Sulf1, Bmp4, Rdh10, Lrp2, Ift57, Alx1
1.35 × 10−7	7	188	Appendage development	Shh, Alx3, Sulf1, Bmp4, Rdh10, Alx1, Alx4
2.49 × 10−8	8	237	Skeletal system morphogenesis	Alx3, Sulf1, Bmp4, Rdh10, Fgfr2, Alx1, Alx4, Ndst1
2.20 × 10−10	10	312	Embryonic organ morphogenesis	Shh, Alx3, Bmp4, Rdh10, Fgfr2, Ift57, Alx1, Six3, Alx4, Ndst1
5.79 × 10−10	10	348	Regionalization	Shh, Bmp4, Lrp2, Disp1, Ift57, Alx1, Cdon, Six3, Alx4, Pgap1
2.20 × 10−10	11	446	Pattern specification proc.	Shh, Alx3, Bmp4, Lrp2, Disp1, Ift57, Alx1, Cdon, Six3, Alx4, Pgap1
1.08 × 10−8	10	478	Embryonic organ development	Shh, Alx3, Bmp4, Rdh10, Fgfr2, Ift57, Alx1, Six3, Alx4, Ndst1
1.76 × 10−11	13	632	Embryonic morphogenesis	Shh, Alx3, Sulf1, Bmp4, Rdh10, Lrp2, Fgfr2, Ift57, Alx1, Cdon, Six3, Alx4, Ndst1
1.80 × 10−8	10	508	Skeletal system development	Shh, Sulf2, Alx3, Sulf1, Bmp4, Rdh10, Fgfr2, Alx1, Alx4, Ndst1
1.61 × 10−10	13	765	Chordate embryonic development	Shh, Sulf2, Alx3, Sulf1, Bmp4, Tcof1, Rdh10, Lrp2, Fgfr2, Ift57, Alx1, Alx4, Ndst1
1.75 × 10−10	13	781	Embryo development ending in birth or egg hatching	Shh, Sulf2, Alx3, Sulf1, Bmp4, Tcof1, Rdh10, Lrp2, Fgfr2, Ift57, Alx1, Alx4, Ndst1
1.11 × 10−7	10	639	Tissue morphogenesis	Shh, Cdc42, Sulf1, Bmp4, Rdh10, Lrp2, Fgfr2, Ift57, Alx1, Six3
1.64 × 10−13	17	1169	Embryo development	Shh, Sulf2, Alx3, Sulf1, Bmp4, Tcof1, Rdh10, Lrp2, Disp1, Fgfr2, Ift57, Alx1, Cdon, Six3, Alx4, Ndst1, Pgap1
1.76 × 10−11	15	1041	Animal organ morphogenesis	Shh, Cdc42, Sulf2, Alx3, Sulf1, Bmp4, Rdh10, Lrp2, Fgfr2, Ift57, Alx1, Cdon, Six3, Alx4, Ndst1
1.24 × 10−7	12	1144	Anatomical structure formation involved in morphogenesis	Shh, Cdc42, Sulf1, Sirt1, Bmp4, Tcof1, Rdh10, Lrp2, Fgfr2, Ift57, Alx1, Cdon
2.49 × 10−8	15	1900	Tissue development	Shh, Cdc42, Sulf2, Sulf1, Sirt1, Bmp4, Tcof1, Rdh10, Lrp2, Fgfr2, Ift57, Alx1, Cdon, Six3, Alx4
GO enrichment (CC)
1.50 × 10−2	2	20	Intraciliary transport particle B	Ift27, Ift57
2.56 × 10−2	2	41	Axonal growth cone	Boc, Lrp2
3.78 × 10−2	3	218	Growth cone	Sirt1, Boc, Lrp2
3.78 × 10−2	3	225	Site of polarized growth	Sirt1, Boc, Lrp2
3.78 × 10−2	4	502	Golgi membrane	Cdc42, Ift27, Ndst3, Ndst1
2.48 × 10−5	11	1449	Golgi apparatus	Shh, Cdc42, Sulf2, Ift27, Sulf1, Lrp2, Ndst3, Ift57, Alx1, Wdr11, Ndst1
1.60 × 10−2	6	926	Cell surface	Shh, Sulf2, Sulf1, Lrp2, Fgfr2, Cdon
GO enrichment (MF)
3.98 × 10−4	2	4	N-acetylglucosamine-6-sulfatase activity	Sulf2, Sulf1
3.98 × 10−4	2	4	[heparan sulfate]-glucosamine N-sulfotransferase activity	Ndst3, Ndst1
4.44 × 10−4	2	5	Arylsulfatase activity	Sulf2, Sulf1
4.63 × 10−2	1	4	Apolipoprotein A-I receptor binding	Cdc42
4.63 × 10−2	1	4	HLH domain binding	Sirt1
4.63 × 10−2	1	4	Keratin filament binding	Sirt1
1.94 × 10−3	2	12	Sulfuric ester hydrolase activity	Sulf2, Sulf1
2.64 × 10−3	2	15	Heparan sulfate sulfotransferase activity	Ndst3, Ndst1
3.98 × 10−4	3	35	Deacetylase activity	Sirt1, Ndst3, Ndst1
3.12 × 10−2	2	61	Sulfotransferase activity	Ndst3, Ndst1
4.63 × 10−2	2	82	Transferase activity, transferring sulphur-containing groups	Ndst3, Ndst1
1.94 × 10−3	4	202	Glycosaminoglycan binding	Shh, Sulf2, Sulf1, Bmp4
1.20 × 10−2	3	152	Carboxylic ester hydrolase activity	Ndst3, Ndst1, Pgap1
2.11 × 10−2	5	742	Hydrolase activity, acting on ester bonds	Sulf2, Sulf1, Ndst3, Ndst1, Pgap1

). Next, we grouped these genes by cellular function and pathway with the KEGG pathway analysis and found that the genes were the most enriched with hedgehog signaling and glycosaminoglycan biosynthesis (Figure 1C and Table 2). Next, we conducted a GO analysis to identify the common biological processes (BPs), cell components (CCs), and molecular functions (MFs) of these genes. We found that the embryonic skeletal system development and embryonic skeletal system morphogenesis were the most enriched with the genes related to frontonasal malformations in BP, the most enriched in terms of the intraciliary transport particle B in CC, and N-acetylglucosamine-6-sulfatase activity and heparan sulfate-glucosamine N-sulfotransferase activity in MF (Figure 1D and Table 2).

Next, we analyzed miRNA target gene regulation using miRTarbase, miRanda, PITA, and TargetScan, and found that miR-338-5p potentially regulates the expression of Boc, Lrp2, Alx1, Sulf1, Sirt1, Cdon, and Zic2; miR-653-5p potentially regulates the expression of Rdh10, Zic2, Pgap1, Sirt1, and Fgfr2; and miR-374c-5p potentially regulates the expression of Alx1, Cdc42, Sirt1, Cdon, and Zic2 (Figure 1E and

Table 3. miRNA prediction for regulating genes related to frontonasal malformations.

miRNA Family	q-Value Bonferroni	q-Value FDR B and H	Hit Count in Query List	Target Gene
miR-338-5p	5.66 × 10−5	1.26 × 10−4	7	Alx1, Boc, Cdon, Lrp2, Sirt1, Sulf1, Zic2
miR-653-5p	8.68 × 10−4	9.98 × 10−3	5	Fgfr2, Pgap1, Rdh10, Sirt1, Zic2
miR-374c-5p	2.95 × 10−2	1.55 × 10−2	5	Alx1, Cdc42, Cdon, Sirt1, Zic2
miR-543-3p	1.15 × 10−2	2.64 × 10−2	4	Fgfr2, Zlc2, Sirt1, Pgap1
miR-124-3p	5.85 × 10−2	2.64 × 10−2	6	Alx1, Cdon, Fgfr2, Shh, Sirt1, Pgap1
miR-6923-5p	1.17 × 10−1	3.38 × 10−2	3	Alx4, Pgap1, Sirt1
miR-3094-3p	1.33 × 10−1	3.49 × 10−2	6	Alx1, Alx4, Cdon, Sirt1, Tcof1, Zic2
miR-6380	2.30 × 10−1	3.77 × 10−2	3	Alx4, Fgfr2, Pgap1
miR-369-3p	2.81 × 10−1	4.17 × 10−2	4	Fgfr2, Pgap1, Sirt1, Sulf1
miR-412-3p	3.16 × 10−1	4.34 × 10−2	3	Cdc42, Ift57, Sulf1

B and H: Benjamini–Hochberg.

).

3.2. Overexpression of miR-338-5p, miR-653-5p, and miR-374c-5p Inhibits Cell Proliferation and Suppresses Expression of Genes Related to Frontonasal Malformations in O9-1 Cells

To test the functional significance of the candidate miRNAs (miR-338-5p, miR-653-5p, and miR-374c-5p) in cell proliferation, we performed cell proliferation assays with a specific mimic for each miRNA in O9-1 cells and found that the overexpression of these miRNAs significantly inhibited cell proliferation (Figure 2A). We confirmed these findings with Bromodeoxyuridine (BrdU) incorporation assays (Figure 2B,C) and immunocytochemical analyses for Ki-67 (Figure 2D,E). There was no apoptotic cell detected in the cells treated with these mimics in TUNEL assays (Figure 2F). Interestingly, we found that inhibitors for these miRNAs did not affect cell proliferation (Figure 2G), suggesting that expression of these miRNAs is relatively low in normal craniofacial development. Therefore, we measured expression of these miRNAs in the developing frontonasal primordium and found that these miRNAs are relatively low-expressed in the frontonasal primordium in C57BL6/J mice at embryonic day E10.5, E11.5, and E12.5 (Figure 2H). Next, to validate the predicted miRNA gene regulation, we conducted qRT-PCR analysis for the genes in O9-1 cells treated with each specific miRNA mimic and inhibitor. We found that the miR-338-5p mimic significantly downregulated expression of Alx1, Lrp2, and Sirt1 in O9-1 cells (Figure 3A); the miR-374c-5p mimic significantly downregulated expression of Alx1, Cdc42, Sirt1, and Zic2 (Figure 3B); and the miR-653-5p mimic significantly downregulated expression of Fgfr2, Pgap1, Rdh10, Sirt1, and Zic2 (Figure 3C). To confirm the dose-dependent effect of the miRNAs on their target genes, the expression of the target genes was analyzed in presence of each miRNA inhibitor. We confirmed that the miR-338-5p inhibitor significantly upregulated expression of Alx1, Lrp2, and Sirt1 (Figure 3D); the miR-374c-5p inhibitor significantly upregulated expression of Alx1, Cdc42, Sirt1, and Zic2 (Figure 3E); and the miR-653-5p inhibitor significantly upregulated expression of Fgfr2, Pgap1, Rdh10, Sirt1, and Zic2 (Figure 3F). Taken together, miR-338-5p, miR-653-5p, and miR-374c-5p can regulate the expression of genes related to frontonasal malformations in a dose-dependent manner.

4. Discussion

miRNAs play crucial roles in the survival of CNC cells during craniofacial development [19], and disruption of miRNA function in these cells results in frontonasal deformities in mice [10,11,12,20]. In this study, we found that overexpression of miR-338-5p, miR-653-5p, and miR-374c-5p inhibited cell proliferation in O9-1 cells through the regulation of genes related to frontonasal malformations. Interestingly, a recent study shows that overexpression of miR-338-5p is related to cleft palate [18]. Therefore, miR-338-5p may be one of the causative miRNAs related to a wide variety of craniofacial developmental defects. The role of miR-653-5p and miR-374c-5p remains largely unknown in both normal development and birth defects. These miRNAs may be upregulated, and therefore the genes crucial for normal development and functions will be downregulated under certain pathological conditions (e.g., cancers and birth defects). For instance, a recent study shows that exosomal miR-653-5p derived from mesenchymal stem cells suppresses laryngeal papilloma progression [21]. In addition, miR-653-5p plays a role in cell proliferation in various cancer cells, such as ovarian cancer [22], papillary thyroid carcinoma [23], breast cancer [24,25,26], gastric cancer [27], and lung cancer [28,29]. Although miR-374c-5p is less well characterized, recent studies show that miR-374c-5p secreted from mesenchymal stem cells inhibits cancer growth and metastasis formation by regulating the epithelial–mesenchymal transition [30,31].

The functions of each miRNA may differ by cell type and timing of expression. Therefore, this study is focused on cranial neural crest cells. In this study, we focused on cell proliferation to study early stages of frontonasal development. During these developmental stages (E10.5–E12.5), osteogenic differentiation is not much involved. In this study, we first curated genes related to frontonasal hypoplasia, frontonasal dysplasia, and hypotelorism and predicted the miRNAs that can regulate these genes. Our findings in regard to miRNA gene regulatory mechanisms will help us understand the potential causes of frontonasal anomalies. In addition, this set of genes will be useful to compare it with genes related to other craniofacial anomalies in order to study spaciotemporal mechanisms in craniofacial development. One of the limitations of this study is that, although we validated the results from bioinformatics with cell culture experiments, these miRNA functions should be evaluated in mouse models with frontonasal anomalies.