MicroRNAs and Gene Regulatory Networks Related to Cleft Lip and Palate

Cleft lip and palate is one of the most common congenital birth defects and has a complex etiology. Either genetic or environmental factors, or both, are involved at various degrees, and the type and severity of clefts vary. One of the longstanding questions is how environmental factors lead to craniofacial developmental anomalies. Recent studies highlight non-coding RNAs as potential epigenetic regulators in cleft lip and palate. In this review, we will discuss microRNAs, a type of small non-coding RNAs that can simultaneously regulate expression of many downstream target genes, as a causative mechanism of cleft lip and palate in humans and mice.


Introduction
Congenital anomalies are a major cause of infant and childhood morbidity, affecting 2-3% of all babies. Cleft lip with/without cleft palate (CL/P) is one of the most prevalent congenital birth defects; it affects 1 in 500 babies in Asian and Native American populations, 1 in 1000 in European-derived populations, and 1 in 2500 in African-derived populations [1]. In the US, cleft lip only (CLO) occurs in 1 in 2800 babies, cleft palate only (CPO) in 1 in 1700 babies, and CL/P in 1 in 1600 babies. A total of 30% of cases of CL/P are syndromic; its etiology is complex with multifactorial effects. For non-syndromic CL/P, it is estimated that 30-50% of cases are caused by genetic factors, and 50-70% are due to non-genetic factors such as abnormal maternal conditions and exposure to teratogens [2][3][4][5]. Individuals with CL/P require multidisciplinary, long-term care from birth to adulthood, with an estimated lifetime cost of more than USD 150,000. Thus, these individuals are affected not only aesthetically and functionally (e.g., at the level of pronunciation, swallowing and suckling), but also economically.
Mice have been frequently used to study craniofacial morphogenesis and its underlying cellular and molecular mechanisms because their developmental processes are similar to those of humans and occur within a short window of time. Given these advantages, genetic mutant mouse models and in vivo cell lineage-tracing methodologies have been used to identify cellular and molecular mechanisms related to CL/P. Upper lip formation begins with enlargement of the maxillary processes (MxPs), which develop from the first pharyngeal arch at the lateral boundary of the stomodeum at embryonic day 9.5 (E9.5) in mice and gestation day 28 in humans [6]. At E10.0 in mice and gestation day 32 in humans, the ventral-lateral ectoderm surface of the frontonasal process (FNP) thickens and forms the nasal placodes (NPs). Around the NPs, the medial and lateral nasal processes (MNPs and LNPs) outgrow in a horseshoe shape, forming the nasal pits. At E10.5 in mice and gestation day 35 in humans, the MxPs show rapid lateral growth and push the nasal pits give rise to fibroblasts in connective tissues, osteoblasts and osteocytes in bones, as well as Schwan cells, which wrap around axons and act as insulators for nerve transmission in the peripheral nervous system. Mesoderm-derived mesenchymal cells give rise to endothelial cells and pericytes in blood capillaries and myoblasts and satellite cells in skeletal muscles. Finally, epithelial cells give rise to basal cells, goblet cells, and ciliated mucous cells in the nasal mucosa, nonkeratinized squamous cells in oral epithelium, and acinar and duct cells in palatal salivary glands (the minor salivary glands located on the palate). Recent advanced technologies, including RNA sequencing at the single-cell level, allow us to identify not only novel cell populations and their fates in development but also cell-type-specific gene regulatory networks for cell specification and function.
works in craniofacial development, as well as histological and functional aspects, are conserved across species. Therefore, genetically modified zebrafish models are widely used to investigate developmental defects, including cleft lip and palate [8,9].
The lip and palate include several cell types derived from CNC cells, mesoderm-derived mesenchymal cells, and epithelial cells (Figure 1). In the palatal shelves, CNC cells give rise to fibroblasts in connective tissues, osteoblasts and osteocytes in bones, as well as Schwan cells, which wrap around axons and act as insulators for nerve transmission in the peripheral nervous system. Mesoderm-derived mesenchymal cells give rise to endothelial cells and pericytes in blood capillaries and myoblasts and satellite cells in skeletal muscles. Finally, epithelial cells give rise to basal cells, goblet cells, and ciliated mucous cells in the nasal mucosa, nonkeratinized squamous cells in oral epithelium, and acinar and duct cells in palatal salivary glands (the minor salivary glands located on the palate). Recent advanced technologies, including RNA sequencing at the single-cell level, allow us to identify not only novel cell populations and their fates in development but also celltype-specific gene regulatory networks for cell specification and function. As stated above, both genetic and environmental factors can contribute to CL/P cases in humans. Several potential non-genetic risk factors have been reported: cigarette smoking [10,11], alcohol consumption [12,13], obesity [14,15], high dietary glycemic index [16], and abnormal nutrient/vitamin conditions [17][18][19]. Moreover, appropriate folic acid supplementation can reduce the risk of developing spina bifida and CL/P in humans [20,21]. It is also known that some chemicals and drugs cause mutagenesis (i.e., they act as muta- As stated above, both genetic and environmental factors can contribute to CL/P cases in humans. Several potential non-genetic risk factors have been reported: cigarette smoking [10,11], alcohol consumption [12,13], obesity [14,15], high dietary glycemic index [16], and abnormal nutrient/vitamin conditions [17][18][19]. Moreover, appropriate folic acid supplementation can reduce the risk of developing spina bifida and CL/P in humans [20,21]. It is also known that some chemicals and drugs cause mutagenesis (i.e., they act as mutagens), but some do not directly induce genetic mutations [22]. Therefore, there is the possibility that some substances may increase or decrease the risk for CL/P through epigenetic mechanisms such as regulation of non-coding RNAs, including microRNAs (miRNAs), transfer RNAs, ribosomal RNAs, small interfering RNAs, and long non-coding RNAs, as well as chromatin modifications such as methylation and acetylation. miRNAs are single-strand non-coding RNAs containing 21-23 nucleotides that can anti-correlatedly and post-transcriptionally regulate the expression of multiple target genes [23][24][25]. miRNAs are transcribed as double-strand pri-miRNA and then cleaved by the DROSHA/DGCR8 complex to generate pre-miRNAs in the nuclei. pre-miRNAs are translocated to the cytoplasm by exportin-5 (XPO5) and cleaved by DICER, an enzyme crucial for miRNA maturation, to form miRNA/mRNA duplexes. Eventually these duplexes attach to Argonaute, a part of the RNA-induced silencing complex (RISC), resulting in loss of one strand and generation of mature miRNAs, which can bind to the 3 -untranslated region (UTR) of the target mRNAs [26,27]. miRNA biogenesis is conserved across species [28]. Importantly, there are multiple binding sites for different miRNAs on the 3 -UTR of the gene; therefore, gene expression is influenced by multiple miRNAs in a spatiotemporal manner. Accumulating evidence indicates that miRNAs play a crucial role in embryogenesis and that altered miRNA expression is associated with various birth defects [29]. In agreement with the importance of miRNAs and their processing enzymes in normal craniofacial development and CL/P in humans [30][31][32][33], mice with a deficiency for Dicer (Dicer F/F ;Wnt1-Cre and Dicer F/F ;Pax2-Cre conditional knockout mice) display severe craniofacial deformities, including cleft palate in both primary and secondary palates [34][35][36]. In zebrafish, mutants homozygous for point mutation dicer1 sa9205 exhibit smaller eyes, craniofacial dysmorphism, and aberrant pigmentation, thus resembling the mouse phenotypes [37].
In the past decade, an increasing number of studies have showed that expression of some miRNAs is drastically altered under pathological conditions [38,39]. These so-called pathogenic miRNAs may suppress genes that are crucial for development and homeostasis, affecting prognosis, drug resistance, and morphogenesis ( Figure 2). Several studies have used RNA-seq to identify miRNA expression during normal lip/palate development as well as in non-syndromic CL/P [40,41]. In addition, mice with loss of function of miRNAs (Dicer1 F/F ;Wnt1-Cre) display severe craniofacial anomalies [35], indicating that some miRNAs are crucial for normal craniofacial development. An increasing number of studies with wild-type mice treated with specific inhibitors for each miRNA may provide some perspective on how an adequate expression of miRNAs is essential for normal orofacial development.

microRNAs Related to Cleft Lip
As of 2022, 55 mouse genes and more than 400 human genes had been reported as related to cleft lip and palate [42,43] in the gene datasets available at CleftGeneDB (https://bioinfo.uth.edu/CleftGeneDB/index.php?csrt=15984704412663399126, accessed on 28 October 2022). Bioinformatic analysis and consequent experimental validation identified miRNA-mediated gene regulatory networks in cleft lip ( Figure 3). For instance,

microRNAs Related to Cleft Palate
An increasing number of studies show that miRNAs are involved in both normal palate and CL/P development in humans and mice ( Figure 4). In our previous studies, we identified five miRNAs that regulate the expression of genes related to cleft lip. These miRNAs have not yet been reported or investigated in embryogenesis and craniofacial development. However, they are suggested to be associated with cancer pathogenesis and prognosis through changes in cell proliferation and differentiation. Since these miRNAs are specifically expressed under specific pathological conditions, such as cancer and cleft lip, they are considered to be pathogenic miRNAs related to cleft lip. Specifically, overexpression of hsa-miR-655-3p and hsa-miR-497-5p inhibits cell proliferation in cultured human lip mesenchymal cells through downregulation of cleft lip-related genes: BCL1, CYPLA1, DMD, FZD6, HOXB3, MID1, NTN, and SATB2 by hsa-miR-655-3p; and BAG4, CHD7, FGFR1, FOXP2, HECTD1, RUNX2, and TFAP2A by hsa-miR-497-5p [43]. hsa-miR-665-3p decreases cell viability by apoptosis or suppresses cell proliferation through downregulation of target genes in various cells, namely BCL2 in human lung adenocarcinoma cells [48], NHEG1 in human neuroblastoma [49], TRIM24 in human castration-resistant prostate cancer [50], and FZD4 in human oral squamous cell carcinoma [51]. In addition, hsa-miR-497-5p inhibits cell proliferation through downregulation of target genes in several human cancer cells, e.g., MAPK1 in cervical cancer cells [52], PDL1 or SLC7A5 in human colorectal cancer cells [53], and WNT3A in human nasopharyngeal carcinoma cells [54]. Thus, miR-124-3p, miR-655-3p, and miR-497-5p may play a key role in cell proliferation as tumor suppressors in cancers and CL/P inducers in development.
The miRNAs described above can commonly inhibit angiogenesis through downregulation of target genes. In fact, miR-205 downregulates VEGA in gastric cancer [71], hepatocellular carcinoma [72], and the extracellular vesicles from diabetic ulcers [73], whereas miR133b in the exosomes secreted from bone marrow mesenchymal stem cells downregulates FBN1 [74] and miR-27b downregulates AMPK in brain microvascular endothelial cells [75], CDH5 (a.k.a. VE-cadherin) in ovarian cancer [76], and VEGFC in gastric cancer [77]. Since angiogenesis is critical for tissue growth and development, these miRNAs may play a role in various tissue processes from morphogenesis through angiogenesis.

microRNAs Related to Cleft Palate
An increasing number of studies show that miRNAs are involved in both normal palate and CL/P development in humans and mice ( Figure 4) Overexpression of miR-374a-5p, miR-4680-3p, and miR-133b suppresses cell proliferation through the regulation of genes related to human cleft palate in cultured human palatal mesenchymal cells: ARNT, BMP2, CRISPLD1, FGFR2, JARID2, MSX1, NOG, RHPN2, RUNX2, WNT5A, and ZNF236 by miR-374a-5p; ERBB2, JADE1, MTHFD1, and WNT5A by miR-4680-3p; and FGFR1, GCH1, PAX7, SMC2 and SUMO1 by miR-133b [78]. Figure 3. Summary of the miRNAs and genes associated with cleft lip in humans and mice. Phenytoin is a known inducer of cleft lip in mice. It inhibits cell proliferation in cultured cells through induction of pathogenic miR-196a-5p, which suppress expression of genes related to cleft lip. CL, cleft lip.

microRNAs Related to Cleft Palate
An increasing number of studies show that miRNAs are involved in both normal palate and CL/P development in humans and mice (Figure 4).   Tapt1  Tbc1d32 Tbx1 Tbx2 Tbx22 Tcof1 Tctn2 Tent5c Tfap2a Tgds Tgfb2 Tgfb3  Tgfbr2 Tgfbr3 Tmem107 Trppc10 Trp53 Trp63 Trps1 Ttc21b Twist1 Ugdh Vax1 Vegfa Wdpcp Wdr19 Wls Wnt5a Wen Zeb1 Zmynd11 Overexpression of miR-374-5p suppresses cell proliferation in several cells: in human non-small cell lung carcinoma cells by suppressing NCK1 expression [80], and in human neural stem cells by suppressing HES1 expression, which promotes neural stem cell differentiation [81]. On the other hand, miR-374-5p shows protective effects in cell viability, reducing apoptotic cell death induced by either oxygen/glucose deprivation (an infant hypoxic-ischemic encephalopathy model) in rat PC12 neuronal cells [82] or by LPS in human pulmonary microvascular endothelial cells [83]. Interestingly, maternal circulating hsa-miR-374-5p is strongly associated with the risk of small-for-gestational-age birth and preterm delivery in humans [84,85], suggesting that miR-374-5p may influence cell proliferation and survival in development.
A total of 44 cleft palate genes are common in humans and mice. A bioinformatic analysis revealed that miR-140-5p is a potential pathogenic miRNA that specifically induces cleft palate in both humans and mice [86]. Overexpression of miR-140-5p suppresses genes that are crucial for palate formation (Pdgfra for the primary palate, Pax9 for the secondary palate, and Bmp2 and Fgf9 for both primary and secondary palate) in human and mouse palatal mesenchymal cells. However, the role of miR-140-5p seems to vary per cell type. Its overexpression induces adipogenic differentiation and lipogenesis through suppression of PDGFRα in pre-adipocytes [87] and alleviates pyroptosis by targeting Ctsb in chondrocytes treated with LPS (an osteoarthritis (OA) model) and in articular cartilage in OA mice [88]. On the other hand, overexpression of miR-140-5p suppresses osteogenic differentiation by targeting SATB2-mediated ERK1/2 and P38MAPK signaling pathways in human vascular smooth muscle cells [89]. Moreover, miR-140-5p binds to NRF2, which is a key molecule for anti-oxidative stress and cellular toxicity, enhances the NRF2/HO-1 signaling pathway, and suppresses cell proliferation, cell migration, and angiogenesis in breast cancer cells under hypoxia conditions [90]. In zebrafish, overexpression of miR-140 results in a cleft between lateral elements of the ethmoid plate, a structural analog of the palate in higher vertebrates, through the suppression of Pdgfra [91]; in mice, miR-140 null mice exhibit submucous cleft palate with hypoplastic palatal bones [92]. Thus, a fine-tuned, precise amount of miR-140 would be crucial for palate development. A single nucleotide polymorphism (SNP) in pre-miR-140 responsible for decreasing miR-140-5p expression is associated with an increased risk of non-syndromic CL/P (nsCL/P) in humans [93]. SNPs in PDGFRA are also associated with risk of developing nsCL/P, with one SNP found at the 3 -UTR near a binding site for miR-140 [94]. These results suggest that the miR-140-PDGFRA axis plays a crucial role in CL/P.
Human linkage analyses suggest that mutations in non-coding miRNA regions are associated with susceptibility to nsCL/P. For instance, miR-152 hypomethylation leading to overexpression is frequently detected in nsCL/P, and overexpression of miR-152 in zebrafish results in craniofacial cartilage dysmorphism [104]. An SNP in rs539075, located in the CDH2 intron where it is suggested to encode miRNAs, is associated with nsCL/P [105]. Mutations in CDH2, which plays a role in EMT, cause syndromic or non-syndromic Peters anomaly, characterized by corneal opacity, hypertelorism, and thin upper lip [106]. Thus, some SNPs are related to the production of miRNAs, while others are related to the binding of miRNAs. For instance, several intronic SNPs located within or near miRNA-binding sites (rs1048201/miR-496 in FGF2, rs3733336/miR-145 in FGF5, and rs546782/miR-187 in FGF9) are suggested to constitute a risk for nsCL/P [107]. rs12532 within the 3 -UTR of MSX1 may affect the binding to miR-3649, leading to a decrease in risk of developing nsCL/P through the regulation of MSX1 expression [108]. Interestingly, miR-let7-3p expression is downregulated in both the plasma from mothers carrying a nsCL/P fetus and lip tissues from nsCL/P individuals [109]. The inhibition of miR-let7-3p suppresses cell proliferation through HHIP upregulation and GLI2 downregulation in human oral keratinocytes. Thus, maternal miR-let-3p expression may become a potential diagnostic biomarker for nsCL/P during pregnancy. Interestingly, expression of miR-378 shows sex differences (i.e., downregulated in female nsCL/P individuals and upregulated in males) [110]. Increasing evidence suggests that maternal miRNA expression and SNPs in miRNA biogenesis enzymes or the 3 -UTR of CL/P-associated genes can be used for screening CL/P during pregnancy. To date, each miRNA-specific inhibitor or mimic, which can modify miRNA expression independently, is developed industrially. Several researchers have succeeded in inducing or rescuing developmental defects by administering these inhibitors/mimics to pregnant mice or zebrafishes. In the near future, these techniques can be applied to repair or reduce the severity of CL/P during pregnancy in humans.

microRNAs Involved in Chemical-Induced Cleft Lip and Cleft Palate
The underlying pathogenic mechanisms in CL/P and CPO are complicated by both genetic and non-genetic factors. Human cohort studies show that maternal exposure to several drugs and chemicals that act as teratogens induces nsCL/P [111,112]. For example, dioxins/TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) [113], phenytoin [114], antibiotics [115], corticosteroids [116], smoking [117], a high dose of alcohol [12,118], and heavy metals [119] are known teratogens for nsCL/P. Human linkage analyses show that mutations in genes related to TCDD metabolism (AHRR, ARNT, and CYP1A1) and a copy number change in AHR are associated with increased risk of CL/P [120,121]. Moreover, mutations in CYP1A1 and GSTT1 in combination with maternal smoking increase the risk of developing CL/P in humans [122,123]. These findings suggest that gene-environment interactions contribute to the pathogenesis, susceptibility, and prevention of CL/P. Non-coding RNAs and methylation status may explain how CL/P-associated gene expression is altered by teratogens. Exposure to several chemicals (e.g., retinoic acid, dexamethasone, dioxins) induces cleft palate in mice and in humans [124][125][126]. Retinoic acid (atRA) induces expression of miR-124-3p [127,128] and miR-106-5p [129] in cultured MEPM cells and the developing palatal shelves in mice. miR-124-3p can inhibit cell proliferation through suppression of genes crucial for palate development, and miR-106-5p induces apoptosis and compromises phosphatidylcholine synthesis/cell membrane synthesis though suppression of Tgfbr2. Importantly, a specific inhibitor for miR-124-3p normalizes cell proliferation under atRA treatments and prevents cleft palate in 65% of atRA-induced cleft palate mice. More recently, another candidate miRNA, miR-340-5p, was identified in atRA-induced cleft palate mice [128]. Therefore, treatment with a combination of miR-124-3p and miR-340-5p inhibitors can prevent cleft palate with almost full penetrance [128]. This suggests that it is possible to prevent CL/P by normalizing maternal pathogenic miRNA expression. Dexamethasone, on the other hand, inhibits cell proliferation through miR-130-3p induction, which suppresses Slc24a2 expression, in cultured MEPM cells [130]. Overexpression or downregulation of miR-130-3p induces or suppresses cell proliferation, migration and invasion, respectively [131,132], whereas its suppression inhibits cell proliferation, TNFα-induced cell migration, and pro-inflammatory cytokine production in MH7A cells (a human rheumatoid arthritis synovial cell line) though upregulation of KLF9 [133].
In summary, modulation of miRNA expression may be key in understanding the toxicity of chemicals and congenital birth defects. In this review, we discussed selected CL/P mouse models and speculated that expression of some miRNAs is commonly altered by exposure to various chemicals. If we can detect these unique pathogenic miRNAs before or during pregnancy, they may become new biomarkers for diagnosis and potential therapeutic targets to prevent or reduce the risk of chemical-related birth defects.

Conclusions
An increasing number of studies suggest a contribution of miRNAs to cleft lip and cleft palate development in humans and mice. Bioinformatic approaches using both sequencing (miRNA-seq and mRNA-seq) and reported cleft-related genes are striking in the identification of miRNAs related to cleft palate. In addition, chemical-induced cleft models can help us identify the underlying mechanisms and allow us to test potential clinical interventions to prevent cleft lip and cleft palate.
Author Contributions: C.I., A.S. and J.I. wrote the paper. All authors reviewed and approved the final version of the manuscript.
Funding: This work was partially supported by grants from the National Institute of Dental and Craniofacial Research (R01DE029818, R01DE026767 to J.I.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Institutional Review Board Statement: Not applicable.

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

Conflicts of Interest:
The authors have declared that no competing interests exist.