The etiology of cleft lip with or without cleft palate (CL/P), a common congenital birth defect, is complex, with genetic and epigenetic, as well as environmental, contributing factors. Recent studies suggest that fetal development is affected by maternal conditions through microRNAs (miRNAs), a group of short noncoding RNAs. Here, we show that miR-129-5p and miR-340-5p suppress cell proliferation in both primary mouse embryonic palatal mesenchymal cells and O9-1 cells, a neural crest cell line, through the regulation of Sox5 and Trp53 by miR-129-5p, and the regulation of Chd7, Fign and Tgfbr1 by miR-340-5p. Notably, miR-340-5p, but not miR-129-5p, was upregulated following all-trans retinoic acid (atRA; tretinoin) administration, and a miR-340-5p inhibitor rescued the cleft palate (CP) phenotype in 47% of atRA-induced CP mice. We have previously reported that a miR-124-3p inhibitor can also partially rescue the CP phenotype in atRA-induced CP mouse model. In this study, we found that a cocktail of miR-124-3p and miR-340-5p inhibitors rescued atRA-induced CP with almost complete penetrance. Taken together, our results suggest that normalization of pathological miRNA expression can be a preventive intervention for CP.

Cleft lip with or without cleft palate (CL/P) is a common congenital birth defect that affects ∼1 in 700 newborns worldwide (Group, 2011). Its etiology is complex, involving genetic and environmental contributing factors, as well as their interactions (Beaty et al., 2016; Dixon et al., 2011). MicroRNAs (miRNAs), which are small non-coding RNAs with 18-25 nucleotides, inhibit the expression of multiple target genes at the post-transcriptional level and are thought to be involved in the development of various diseases (Huang et al., 2020). Previous studies suggest that these molecules play a crucial role in craniofacial development (Mukhopadhyay et al., 2019; Seelan et al., 2014; Warner et al., 2014; Xu et al., 2018, 2021b), and growing evidence from single nuclear polymorphism studies suggests that miRNAs are involved in nonsyndromic CL/P in humans (Li et al., 2016). However, it remains unclear how miRNAs contribute specifically to CL/P.

Currently, a significant association with nonsyndromic CL/P has been reported for pre-miR-140 (Li et al., 2010), pre-miR-146a (Pan et al., 2018), miR-140 (Li et al., 2011), miR-149 (Stussel et al., 2022), miR-378 (Xu et al., 2021a) and DROSHA (Xu et al., 2018). Moreover, complete loss of mature miRNAs leads to CP in mice with a conditional deletion of Dicer1 in cranial neural crest (CNC) cells (Wnt1-Cre;Dicer1F/F and Pax2-Cre;Dicer1F/F) (Barritt et al., 2012; Nie et al., 2011; Zehir et al., 2010). Furthermore, mice deficient for the miR-17-92 cluster exhibit cleft lip (CL) and cleft palate (CP) through upregulation of Fgf10, Tbx1 and Tbx3 (de Pontual et al., 2011,b7; Wang et al., 2013), and mice overexpressing the inhibitory miR-17-92 (PMIS-Ics-miR17-92 mice and PMIS-Ics-miR17-18) exhibit CP (Cao et al., 2016; Ries et al., 2017). By contrast, upregulation of several miRNAs has been reported in CP. For example, overexpression of miR-140 causes CP through dysregulation of platelet-derived growth factor receptor α (PDGFRα) signaling in mouse embryonic palatal mesenchymal (MEPM) cells, zebrafish and humans (Eberhart et al., 2008; Li et al., 2020; Rattanasopha et al., 2012). Our previous studies have shown that a total of 18 miRNAs are potentially involved in the regulation of genes related to CP (Suzuki et al., 2018a,b). Among them, miR-124-3p expression is induced by excessive maternal exposure to all-trans retinoic acid (atRA; tretinoin), a derivative of vitamin A, leading to CP in mice due to inhibition of cell proliferation in the developing palate. In addition, administration of a miR-124-3p inhibitor rescues the CP phenotype in 65% of the atRA-induced CP mice (Yoshioka et al., 2021a). Nonetheless, it remains unknown whether any combinations of treatments can prevent CP more efficiently. This study therefore aimed to improve the success rate of the rescue of the CP phenotype in an atRA-induced CP mouse model.

Overexpression of miR-129-5p and miR-340-5p inhibits cell proliferation in MEPM and O9-1 cells

Our recent bioinformatic study highlighted several gene regulatory networks, including miR-129-5p and miR-340-5p, that play crucial roles in craniofacial development (Yan et al., 2020); however, their roles in palatogenesis have not yet been determined. To investigate the contribution of these miRNAs to orofacial clefts in palatal mesenchymal cells, we first treated primary MEPM cells, primary mouse embryonic lip mesenchymal (MELM) cells and O9-1 cells, an established mouse neural crest cell line, with either a miR-129-5p or miR-340-5p mimic. We found that miR-129-5p and miR-340-5p mimics significantly inhibited cell proliferation in MEPM and O9-1 cells, but not in MELM cells (Fig. 1A,B and Fig. S1A), indicating that these miRNAs affect the growth of the palatal shelves but not lip formation. Next, to identify target genes regulated by miR-129-5p and miR-340-5p, we performed quantitative RT-PCR analysis for genes associated with CP in MEPM and O9-1 cells. The candidate genes were selected based on their expression, on miRNA binding site prediction (Fig. S2) and on association with CP: genes differentially expressed between E10.5 and E11.5, E11.5 and E12.5, E12.5 and E13.5, and E13.5 and E14.5 (Yan et al., 2020), and genes reported to be CP-related genes (Suzuki et al., 2018a,b). We found that Egfr (E10.5 versus E11.5 and E11.5 versus E12.5), Fgf18 (E11.5 versus E12.5), Hic1, Lhx8, Sox5, Tfap2a, Trp53, Twist1 and Zeb1 (E13.5 versus E14.5) were candidate genes regulated by miR-129-5p, and that Chd7, Fign and Tgfbr1 (E10.5 versus E11.5), and Met (E12.5 versus E13.5) were candidate genes regulated by miR-340-5p. Among them, overexpression of miR-129-5p significantly downregulated expression of Sox5, Tfap2a, Trp53 and Zeb1 in MEPM cells (Fig. 1C), and of Egfr, Hic1 and Trp53 in O9-1 cells (Fig. S1B), whereas overexpression of miR-340-5p significantly downregulated the expression of Chd7, Fign and Tgfbr1 in MEPM cells (Fig. 1D), and of Chd7, Fign, Met and Tgfbr1 in O9-1 cells (Fig. S1C). Next, to evaluate the effect of loss of function of each miRNA, we conducted cell proliferation assays with each miRNA inhibitor in MEPM cells. We observed no effect on cell proliferation in cells treated with these miRNA inhibitors, although suppression of miR-129-5p and miR-340-5p significantly upregulated the expression of Egfr, Hic1, Lhx8, Sox5, Trp53 and Twist1, and of Chd7, Fign, Met, and Tgfbr1, respectively, in MEPM cells (Fig. 1E-G). Similarly, suppression of miR-129-5p and miR-340-5p significantly upregulated expression of Egfr, Fgf18, Hic1 and Trp53, and of Chd7, Fign and Tgfbr1, respectively, in O9-1 cells without affecting proliferation (Fig. S1D,E,F). This suggests that, although some target genes may be involved in cell proliferation, either directly or indirectly, upregulated expression of these genes might not further promote cell proliferation as cell proliferation is very active during embryogenesis. However, expression of Sox5, Hic1 and Trp53, and of Chd7, Fign and Tgfbr1, are regulated in a dose-dependent manner by miR-129-5p and miR-340-5p, respectively, and, importantly, the expression of the target genes was specifically regulated (Fig. 1C,D,F,G).

Fig. 1.

Overexpression of miR-129-5p and miR-340-5p suppresses cell proliferation and gene expression in MEPM and MELM cells. (A,B) Cell proliferation assays using MEPM cells (A) or MELM cells (B) treated with a control, a miR-129-5p or a miR-340-5p mimic. Two-way ANOVA with Dunnett's test (n=6). **P<0.01 versus control. (C,D) Quantitative RT-PCR for the indicated genes after treatment with a control or a miR-129-5p mimic (C), or a control or a miR-340-5p mimic (D). Multiple t-tests adjusted by Bonferroni (n=6). **P<0.01 and ***P<0.001 versus control. (E) Cell proliferation assays using MEPM cells treated with a control, a miR-129-5p or a miR-340-5p inhibitor (n=6). (F,G) Quantitative RT-PCR for the indicated genes after treatment with a control or a miR-129-5p inhibitor (F), or a control or a miR-340-5p inhibitor (G). Multiple t-tests adjusted by Bonferroni (n=6). **P<0.01 and ***P<0.001 versus control.

Fig. 1.

Overexpression of miR-129-5p and miR-340-5p suppresses cell proliferation and gene expression in MEPM and MELM cells. (A,B) Cell proliferation assays using MEPM cells (A) or MELM cells (B) treated with a control, a miR-129-5p or a miR-340-5p mimic. Two-way ANOVA with Dunnett's test (n=6). **P<0.01 versus control. (C,D) Quantitative RT-PCR for the indicated genes after treatment with a control or a miR-129-5p mimic (C), or a control or a miR-340-5p mimic (D). Multiple t-tests adjusted by Bonferroni (n=6). **P<0.01 and ***P<0.001 versus control. (E) Cell proliferation assays using MEPM cells treated with a control, a miR-129-5p or a miR-340-5p inhibitor (n=6). (F,G) Quantitative RT-PCR for the indicated genes after treatment with a control or a miR-129-5p inhibitor (F), or a control or a miR-340-5p inhibitor (G). Multiple t-tests adjusted by Bonferroni (n=6). **P<0.01 and ***P<0.001 versus control.

Inhibitors of miR-124-3p and miR-340-5p rescue decreased cell proliferation in atRA-treated palatal mesenchymal cells

Treatment with atRA inhibited the proliferation of MEPM and O9-1 cells (Fig. 2A and Fig. S3A). We found that expression of miR-340-5p, but not miR-129-5p, was specifically upregulated following atRA treatment (Fig. 2B and Fig. S3B). To evaluate the contribution of miR-129-5p and miR-340-5p to cell proliferation, we treated MEPM and O9-1 cells with either a miR-129-5p or miR-340-5p inhibitor, under atRA treatment conditions, and found that the miR-340-5p inhibitor partially normalized the proliferation of MEPM and O9-1 cells (Fig. 2C and Fig. S3C); by contrast, the miR-129-5p inhibitor failed to rescue the suppressed cell proliferation, suggesting that it is not involved in atRA-induced CP. As expected, the expression of Chd7, Fign and Tgfbr1 was partially restored by the miR-340-5p inhibitor under atRA treatment conditions (Fig. 2D and Fig. S3D).

Fig. 2.

Normalization of miR-340-5p and miR-124-3p expression restores the atRA-induced cell proliferation defect in MEPM cells. (A) Cell proliferation assays in MEPM cells treated with 10 µM atRA for 24, 48 and 72 h. Two-way ANOVA with Dunnett's test (n=6). ***P<0.001 versus control. (B) Quantitative RT-PCR for miR-129-5p or miR-340-5p after treatment with atRA for 24 h in MEPM cells. Multiple t-tests adjusted by Bonferroni (n=6). **P<0.01 and ***P<0.001 versus control. (C) Cell proliferation assays in MEPM cells treated with a control, a miR-124-3p, a miR-129-5p or a miR-340-5p inhibitor under atRA treatment conditions for 0, 24, 48 or 72 h (n=6). ***P<0.001 versus control. ##P<0.01 versus atRA. (D) Quantitative RT-PCR for the indicated genes in MEPM cells after treatment with a miR-340-5p inhibitor for 24 h under atRA treatment conditions. One-way ANOVA with Tukey's test (n=6). ***P<0.001 versus control. ##P<0.01 and ###P<0.001 versus atRA. (E) Cell proliferation assays in MEPM cells treated with a control, a miR-340-5p inhibitor or a cocktail of miR-124-3p and miR-340-5p inhibitors under atRA treatment conditions for 0, 24, 48 or 72 h (n=6). ***P<0.001 versus control. ##P<0.01 and ###P<0.001 versus atRA. (F) BrdU staining in MEPM cells after treatment with or without a miR-340-5p inhibitor and/or miR-124-3p inhibitor under atRA treatment conditions for 72 h. One-way ANOVA with Tukey's test (n=8-10). **P<0.001 versus control, ##P<0.01 versus atRA. Scale bars: 50 μm. (G) Quantitative RT-PCR for the indicated genes in MEPM cells after treatment with a control, a miR-124-3p inhibitor, a miR-340-5p inhibitor or a cocktail of miR-124-3p and miR-340-5p inhibitors for 24 h under atRA treatment conditions. One-way ANOVA with Tukey's test (n=6). **P<0.01 and ***P<0.001 versus control. ##P<0.01 and ###P<0.001 versus atRA. ns, not significant.

Fig. 2.

Normalization of miR-340-5p and miR-124-3p expression restores the atRA-induced cell proliferation defect in MEPM cells. (A) Cell proliferation assays in MEPM cells treated with 10 µM atRA for 24, 48 and 72 h. Two-way ANOVA with Dunnett's test (n=6). ***P<0.001 versus control. (B) Quantitative RT-PCR for miR-129-5p or miR-340-5p after treatment with atRA for 24 h in MEPM cells. Multiple t-tests adjusted by Bonferroni (n=6). **P<0.01 and ***P<0.001 versus control. (C) Cell proliferation assays in MEPM cells treated with a control, a miR-124-3p, a miR-129-5p or a miR-340-5p inhibitor under atRA treatment conditions for 0, 24, 48 or 72 h (n=6). ***P<0.001 versus control. ##P<0.01 versus atRA. (D) Quantitative RT-PCR for the indicated genes in MEPM cells after treatment with a miR-340-5p inhibitor for 24 h under atRA treatment conditions. One-way ANOVA with Tukey's test (n=6). ***P<0.001 versus control. ##P<0.01 and ###P<0.001 versus atRA. (E) Cell proliferation assays in MEPM cells treated with a control, a miR-340-5p inhibitor or a cocktail of miR-124-3p and miR-340-5p inhibitors under atRA treatment conditions for 0, 24, 48 or 72 h (n=6). ***P<0.001 versus control. ##P<0.01 and ###P<0.001 versus atRA. (F) BrdU staining in MEPM cells after treatment with or without a miR-340-5p inhibitor and/or miR-124-3p inhibitor under atRA treatment conditions for 72 h. One-way ANOVA with Tukey's test (n=8-10). **P<0.001 versus control, ##P<0.01 versus atRA. Scale bars: 50 μm. (G) Quantitative RT-PCR for the indicated genes in MEPM cells after treatment with a control, a miR-124-3p inhibitor, a miR-340-5p inhibitor or a cocktail of miR-124-3p and miR-340-5p inhibitors for 24 h under atRA treatment conditions. One-way ANOVA with Tukey's test (n=6). **P<0.01 and ***P<0.001 versus control. ##P<0.01 and ###P<0.001 versus atRA. ns, not significant.

Our previous study showed that the miR-124-3p inhibitor can partially normalize cell proliferation under atRA treatment conditions in MEPM cells (Yoshioka et al., 2021a). We thus hypothesized that a combination of miR-124-3p and miR-340-5p could rescue the cell proliferation defect more efficiently. To evaluate the efficiency of this combination, we treated MEPM and O9-1 cells with a cocktail of miR-124-3p and miR-340-5p inhibitors, and found that the cocktail of inhibitors completely rescued the suppressed cell proliferation phenotype under atRA conditions (Fig. 2E,F and Fig. S4A,B). In addition, there was no additive effect on the expression of target genes; Fst, Vcan and Zeb1 were regulated by miR-124-3p and Chd7, Fign and Tgfbr1 were regulated by miR-340-5p (Fig. 2F,G and Fig. S4C), indicating that the expression of these target genes was specifically regulated by each miRNA and not altered by another miRNA. We also confirmed that knocking down these genes inhibited cell proliferation (Fig. S5).

atRA upregulates miR-340-5p expression in the developing palate, and normalization of the expression of miR-124-3p and miR-340-5p rescues atRA-induced CP in mice

Oral administration of atRA to pregnant mice is known to induce CP (Yoshioka et al., 2021a). To examine the contribution of miR-129-5p and miR-340-5p to CP development, we analyzed their expression in the palatal shelves of mice treated with atRA or vehicle. The expression of miR-340-5p was detectable through E12.5 to E14.5 and significantly upregulated upon atRA administration (Fig. 3A). By contrast, the expression of miR-129-5p was not detectable at E12.5 and E13.5 in mice treated with either atRA or vehicle. At E14.5, it was slightly expressed [miR-129-5p per miR-26a-5p (a housekeeping miRNA) expression: 6.12±0.58×10−4] but downregulated (miR-129-5p per miR-26a-5p expression: 3.79±0.32×10−4) with atRA treatment, indicating that miR-129-5p is not responsible for atRA-induced CP. Next, to evaluate the role of miR-340-5p in CP, we treated atRA-induced CP mice with the miR-340-5p inhibitor; as expected, this inhibitor prevented CP in 47% of the mice (Fig. 3B). Our previous study showed that normalization of miR-124-3p expression rescues atRA-induced CP in 65% of mice (Yoshioka et al., 2021a). We therefore hypothesized that normalization of expression of both miR-340-5p and miR-124-3p ameliorates their susceptibility to atRA and prevents CP. miR-124-3p was significantly upregulated by atRA treatment in the palatal shelves of E13.5 and E14.5 mice (Fig. S6A). Interestingly, genes targeted by miR-340-5p were downregulated by atRA treatment at E12.5 to E14.5 (Fig. S6B). By contrast, the expression of target genes of miR-124-3p was downregulated at E13.5 and E14.5 (Fig. S6B). These results suggest that each miRNA differently contributes to palate development at different developmental stages. To test this hypothesis, we administered a cocktail of miR-124-3p and miR-340-5p inhibitors to atRA-induced CP mice. Notably, the combination of these inhibitors prevented CP in 96% of the mice (Fig. 3B) through normalization of cell proliferation (Fig. 3C) and target gene expression (Fig. 3D). We further analyzed the expression level and pattern of these molecules and found that expression compromised by atRA treatment was restored with a combination of these inhibitors (Fig. S7). Limb abnormalities induced by atRA (Collins et al., 2006; Shimizu et al., 2007) were also rescued with the inhibitors (Fig. S8). However, although we observed that the miRNA cocktail normalized CP and limb defects, the embryos still failed to survive after birth. As we tested the miRNA cocktail that specifically inhibits miRNAs induced by atRA in the developing palate, we do not know whether major organs would be rescued by the treatment.

Fig. 3.

Normalization of miR-340-5p expression partially rescues atRA-induced cleft palate, and normalization of miR-124-3p and miR-340-5p expression rescues atRA-induced cleft palate in mice. (A) Quantitative RT-PCR for miR-340-5p in the palatal shelves of atRA-induced cleft palate mice at E12.5, E13.5 and E14.5 (n=6). **P<0.01 and ***P<0.001 versus control at each indicated day. (B) Hematoxylin and Eosin staining of the face of E18.5 C57BL/6J mice treated with control vehicle, atRA, atRA+miR-340-5p inhibitor, or atRA+miR-124-3p+miR-340-5p inhibitors at the anterior, middle and posterior of the secondary palate. Scale bars: 500 μm. (C) BrdU staining in the developing palate of E13.5 C57BL/6J mice treated with control inhibitor, with atRA, with atRA and miR-340-5p inhibitor, or with atRA, miR-340-5p and miR-124-3p inhibitors. One-way ANOVA with Tukey's test (n=10-12). ***P<0.001 versus control. ###P<0.001 versus atRA. (D) Quantitative RT-PCR for the indicated genes in atRA-induced cleft palate mice after treatment with control inhibitor, miR-124-3p inhibitor, miR-340-5p inhibitor, or a cocktail of miR-124-3p and miR-340-5p inhibitors. One-way ANOVA with Tukey's test (n=6). **P<0.01 and ***P<0.001 versus control. #P<0.05, ##P<0.01 and ###P<0.001 versus atRA. ns, not significant.

Fig. 3.

Normalization of miR-340-5p expression partially rescues atRA-induced cleft palate, and normalization of miR-124-3p and miR-340-5p expression rescues atRA-induced cleft palate in mice. (A) Quantitative RT-PCR for miR-340-5p in the palatal shelves of atRA-induced cleft palate mice at E12.5, E13.5 and E14.5 (n=6). **P<0.01 and ***P<0.001 versus control at each indicated day. (B) Hematoxylin and Eosin staining of the face of E18.5 C57BL/6J mice treated with control vehicle, atRA, atRA+miR-340-5p inhibitor, or atRA+miR-124-3p+miR-340-5p inhibitors at the anterior, middle and posterior of the secondary palate. Scale bars: 500 μm. (C) BrdU staining in the developing palate of E13.5 C57BL/6J mice treated with control inhibitor, with atRA, with atRA and miR-340-5p inhibitor, or with atRA, miR-340-5p and miR-124-3p inhibitors. One-way ANOVA with Tukey's test (n=10-12). ***P<0.001 versus control. ###P<0.001 versus atRA. (D) Quantitative RT-PCR for the indicated genes in atRA-induced cleft palate mice after treatment with control inhibitor, miR-124-3p inhibitor, miR-340-5p inhibitor, or a cocktail of miR-124-3p and miR-340-5p inhibitors. One-way ANOVA with Tukey's test (n=6). **P<0.01 and ***P<0.001 versus control. #P<0.05, ##P<0.01 and ###P<0.001 versus atRA. ns, not significant.

An accumulating number of studies show that miRNAs and exosome-derived miRNAs play crucial roles in various diseases and in embryogenesis, and are thus considered to be diagnostic biomarkers and therapeutic targets. As miRNAs can regulate multiple genes at the same time, the effect of one miRNA is broader than the impact on one gene. It was recently shown that a combination of miRNAs is a more effective treatment for diseases and tissue regeneration (Miroshnichenko and Patutina, 2019). For example, a combination of a miR-199a/b-3p mimic and miR-10b inhibitor suppresses hepatocellular carcinoma proliferation and tumor growth in vitro and in vivo (Shao et al., 2020). In addition, pre-treatment with a miR-181a/-212 mimic inhibits tumor growth in a mouse model for rhabdomyosarcoma (Pozzo et al., 2021), and pre-treatment with a pre-miR-21/-24/-221 cocktail is capable of improving the survival rate of engrafted cardiac progenitor cells in ischemic heart disease (Hu et al., 2011).

miR-340 has been associated with multiple biological processes, including cell proliferation, migration and apoptosis (Wu et al., 2011). miR-340-3p and miR-340-5p act as negative regulators of tumor growth and metastasis through the suppression of target genes (Wang et al., 2020; Wu et al., 2011), and suppression of miR-340-5p inhibits tumor growth through suppression of Bmp4 in a xenograft mouse model (Zhao et al., 2018). Therefore, the expression of miR-340-5p is involved in cancer prognosis and growth. Interestingly, miR-340-5p is expressed in craniofacial tissues, and its levels increase in the plasma of individuals with CP (Li et al., 2016). In this study, we found that miR-340-5p regulates Chd7, Fign and Tgfbr1 in MEPM and O9-1 cells in a dose-dependent manner. CHD7 is a member of the chromodomain helicase DNA-binding domain family of ATP-dependent chromatin remodeling enzymes; mutations in CHD7 cause CHARGE syndrome in humans, and mice with heterozygous mutations in Chd7 display CP with 35% penetrance (Bosman et al., 2005). FIGN, an ATP-dependent microtubule severing enzyme, is involved in cellular microtube array and mitosis (Mukherjee et al., 2012). Fign is expressed in the first branchial arch, optic vesicle, pelvic anlage and calvaria (Cox et al., 2000), and Fign null mice exhibit developmental defects, including CP, microphthalmia, craniosynostosis, polydactyl and pelvic girdle dysgenesis (Cox et al., 2000; Yang et al., 2006). Finally, TGFBR1 (also known as ALK5), a receptor of TGFβ, exerts a wide spectrum of biological functions, and mice with conditional null mutations for Tgfbr1 (K14-cre;Tgfbr1F/F and Wnt1-cre;Tgfbr1F/F) exhibit craniofacial defects, including CP (Dudas et al., 2006). However, CP penetrance in these mutant mice varies, with 65% in Adamts20−/−;Vcan+/− (Enomoto et al., 2010), 100% in Zeb1−/− (Gong et al., 2000; Takagi et al., 1998), 100% in Wnt1-Cre;Tgfbr1F/F (Dudas et al., 2006), 50% in Fign−/− (Yang et al., 2006) and 35% in carriers of Chd7 point mutations (Bosman et al., 2005), suggesting that cell proliferation is regulated by multiple genes with different timings and contributions. atRA-induced CP rescue experiments in these mutant mice will be useful to evaluate the crucial nature of these genes. In addition, cells with a loss of gene of interest (e.g. via CRISPR/Cas9) will help delineate the roles of specific genes in cell proliferation. These future studies will substantially increase the understanding of miR-mediated cell proliferation regulation.

Our results show that knocking down these genes suppresses cell proliferation to a different degree in MEPM cells. Moreover, cell proliferation suppressed with atRA is restored by co-suppression of miR-124-3p and miR-340-5p, whereas co-suppression has no additive effect on gene expression compared with treatment with each individual inhibitor. Interestingly, in keloid fibroblasts, the long non-cording RNA HOXA11-AS can downregulate miR-124-3p expression, which leads to upregulation of TGFBR1 expression through binding at the 3′-UTR, resulting in activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway, which induces apoptosis in fibroblasts, inhibits angiogenesis and promotes keloid formation (Jin et al., 2020). Thus, miR-340-5p and miR-124-3p potentially target both Zeb1 and Tgfbr1 in some cell types; however, the co-regulation of Zeb1 and Tgfbr1 by miR-124-3p and miR-340-5p may be a cell-specific and/or disease-dependent phenomenon.

In summary, our results show that miR-340-5p plays crucial roles in atRA-induced CP through regulation of Chd7, Fign and Tgfbr1. As the genes regulated by miR-340-5p were identified at an early stage of palate development, miR-340-5p may be crucial for the initiation of CNC-derived mesenchymal cell proliferation. On the other hand, the genes regulated by miR-124-3p were identified at E13.5 and E14.5; therefore, miR-124-3p may regulate both proliferation and differentiation during palate formation. Thus, a cocktail of miR-124-3p and miR-340-5p inhibitors might improve the entire process of palate formation.

Cell culture

Primary MEPM cells were isolated from the palatal shelves of C57BL/6J mice at E13.5, and primary MELM cells were isolated from the anterior half of the maxillary process, a developing lip region, of C57BL/6J mice at E11.5, as previously described (Yoshioka et al., 2021a,b). MEPM and MELM cells were maintained with Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin (Sigma-Aldrich), 2-mercaptoethanol (Gibco) and nonessential amino acids (Sigma-Aldrich) at 37°C in a humidified atmosphere with 5% CO2. The O9-1 mouse cranial neural crest cell line (SCC049, Sigma-Aldrich) was cultured with conditioned medium provided from the STO mouse embryonic fibroblast cell line (CRL-1503, ATCC), as previously described (Suzuki et al., 2019).

Animals

C57BL/6J mice were obtained from The Jackson Laboratory. Pregnant female mice were orally administered a single dose of 50 mg/kg atRA (R2625, Sigma-Aldrich) suspended in 10% ethanol and 90% corn oil emulsion at E11.5. Control mice received an equivalent amount of emulsion without atRA (0.1 ml/10 g body weight) (n=6 per group). For the rescue experiments, 50 mg/kg atRA was orally administered at E11.5, and the miR-340-5p inhibitor and/or miR-124-3p inhibitor (Integrated DNA technologies) was then intraperitoneally injected at 5 mg/kg at E12.5 and E13.5 (n=6 per group). The protocol was approved by the Animal Welfare Committee (AWC) and the Institutional Animal Care and Use Committee (IACUC) of UT Health (AWC 19-0079). All mice were maintained at the animal facility of UT Health.

Cell proliferation assay

Cells were plated onto 96-well cell culture plates at a density of 5000 cells (MEPM and MELM) and 1000 cells (O9-1) per well and treated with a mimic for negative control (4464061), miR-129-5p (4464066; MC10195) and miR-340-5p (4464066; MC12670) [mirVana miRNA mimic (chemically modified double-stranded RNA molecules), Thermo Fisher Scientific]; or an inhibitor for negative control (4464079), miR-129-5p (4464084; MH10195), miR-340-5p (4464084; MH12670) and miR-124-3p (4464084; MH10691) [mirVana miRNA inhibitor (chemically modified, single-stranded oligonucleotides with patented secondary structure) Thermo Fisher Scientific]; or a siRNA for negative control (SIC001, Millipore Sigma), Chd7 (SASI_Mm02_00298181, SASI_Mm02_00298183 and SASI_Mm02_00298184), Fign (SASI_Mm01_00168509, SASI_Mm01_00168512 and SASI_Mm02_00326406), Tgfbr1 (SASI_Mm01_00169048, SASI_Mm01_00169049 and SASI_Mm01_00169052), Vcan (SASI_Mm02_00296627, SASI_Mm02_00296628 and SASI_Mm02_00296629) and Zeb1 (SASI_Mm02_00320896, SASI_Mm01_00144364, and SASI_Mm01_00144365) using the Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific) according to the manufacturer's protocol (3 pmol of mimic, inhibitor or siRNA and 0.3 µl of transfection reagent in 100 µl DMEM per well). Cell proliferation was measured using the Cell Counting Kit 8 (Dojindo Molecular Technologies) 24, 48 or 72 h after treatment with either mimics or inhibitors (n=6 per group). For the atRA exposure experiments, MEPM cells or O9-1 cells were plated onto 96-well cell culture plates at a density of 5000 cells (MEPM) and 1000 cells (O9-1) per well, and treated with 10 μM atRA. After 24 h of atRA treatment, cells were treated with the inhibitors; after 24, 48 or 72 h of treatment, cell numbers were determined as described above.

Quantitative RT-PCR

MEPM and O9-1 cells were plated onto a 60 mm dish at a density of 40,000 cells per dish. When the cells reached 80% confluence, they were treated with a mimic or inhibitor for miR-129-5p, miR-340-5p, miR-124-3p or negative control at 3 pmol in 6 µl of transfection reagent (Lipofectamine RNAiMAX transfection reagent in 4 ml DMEM per dish). After 24 h of treatment, total RNA was extracted with the QIAshredder and miRNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. For the atRA experiments, the cells were plated onto a 60 mm dish at a density of 50,000 cells per dish and treated with 10 μM atRA for 24 h. Total RNA from the treated cells (n=6 per group) was extracted and mRNA expression level was measured by quantitative RT-PCR, as previously described (Yoshioka et al., 2021b). For the animal experiments, the palatal shelves were micro-dissected at E12.5-E14.5, and total RNA was extracted for qRT-PCR analysis; the PCR primers used in this study are listed in Table S1. The expression level of each mRNA was normalized to Gapdh. miRNA expression was measured with Taqman Fast Advanced Master Mix and Taqman Advanced miR cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Probes for miR-124-3p (mmu480901_mir), miR-129-5p (mmu480913_mir), miR-340-5p (mmu481072_mir) and miR-26a-5p (477995_mir) were purchased from Thermo Fisher Scientific. The expression level of each miRNA was normalized by a housekeeping reference miRNA: miR-26a-5p.

BrdU incorporation assay

MEPM cells were plated onto 35 mm dishes at a density of 25,000/dish and treated with 10 μM atRA, or control vehicle (dimethyl sulfoxide) (n=3 per group). After 72 h, the cells were incubated with 100 μg/ml BrdU (Sigma-Aldrich) for 1 h. BrdU (100 µg/g body weight) was intraperitoneally injected to pregnant mice at E13.5 (n=6 per group) and tissues were collected for analysis after 1 h. Incorporated BrdU was detected by a rat monoclonal antibody against BrdU (ab6326; Abcam, 1:1000), as previously described (Yoshioka et al., 2021a). BrdU-positive cells were counted at 8-12 different areas for each independent experiment.

Histological analysis

Embryos treated with atRA and miRNA inhibitors or control inhibitors were collected at E13.5 and E18.5, and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) overnight at 4°C. The samples from E18.5 embryos were decalcified with 10% ethylenediamine tetra acetic acid disodium-dihydrate salt for 1 month at 4°C. All samples were embedded into paraffin wax and serial coronal sections were cut at 4 µm for further analyses. Hematoxylin and Eosin staining was performed as previously described (Yoshioka et al., 2021a). For immunohistochemistry, deparaffinized sections were treated with citrate buffer (pH 6.0) at 95°C for 30 min for antigen retrieval, followed by 0.3% hydrogen peroxide in methanol for endogenous peroxidase blocking. The sections were incubated with a rabbit polyclonal antibody for CHD7 (1:100, NBP1-77393, Novus Biological), FIGN/fidgetin (1:100, ab232756, Abcam), TGF-β receptor type I (1:100, ab235178, Abcam), versican (1:100, NBP1-85432, Novus Biologicals) or ZEB1 (1:100, NBP1-05987, Novus Biologicals) overnight at 4°C. The sections were then incubated with a secondary antibody, biotinylated goat anti-rabbit IgG (H+L) (1:500, BA-1000; Vector Laboratories), for 1 h at room temperature, followed by the Vectastain ABC horseradish peroxidase kit (PK4000, Vector Laboratories) for 1 h at room temperature and then DAB (SK-4105, Vector Laboratories) for final visualization. Fluorescence images were obtained using a confocal microscope (Ti-C2, Nikon) and color images were obtained using a light microscope (BX43, Olympus). n=6 per group in each experiment.

Prediction of miRNA target genes

To predict the miRNA target genes, miRNA-binding sites were analyzed using STarMir (Kanoria et al., 2016), Targetscan (http://www.targetscan.org/vert_80/) (19167326), miRDB (http://mirdb.org/mirdb/index.html) (Wong and Wang, 2015) and miRBase (https://www.mirbase.org/) in the 3′UTRs of the candidate genes. Murine genome sequences (Build 38) were obtained from the UCSC genome browser (https://genome.ucsc.edu/). When there were multiple 3'UTR-seed sites, the site with high logistic probability, which is a measure of the predicted reliability of the site, was used.

Statistical analysis

All experiments were performed independently three times, and all statistical analyses were performed using the SPSS software (version 28.0, IBM). The statistical significance of any difference between multiple groups was evaluated using multiple unpaired two-tailed t-tests and adjusted by Bonferroni. For a comparison between multiple groups with one factor, a one-way analysis of variance (ANOVA) with Tukey's honest significant difference test was used for assessment. Cell proliferation assays were analyzed using a two-way ANOVA with Dunnett's test or Tukey's honest significant difference test. An adjusted P<0.05 was considered to be statistically significant. Data are mean±s.d. in the graphs.

We thank Junbo Shim and Yurie Mikami for technical assistance.

Author contributions

Conceptualization: J.I.; Methodology: H.Y., A.S., C.I., J.I.; Formal analysis: H.Y., C.I.; Investigation: A.S.; Writing - original draft: A.S., J.I.; Writing - review & editing: H.Y., A.S., J.I.; Visualization: J.I.; Supervision: J.I.; Funding acquisition: J.I.

Funding

This study was supported by grants from the National Institute of Dental and Craniofacial Research (National Institutes of Health) (R01DE029818, R03DE026208, R03DE026509 and R01DE026767 to J.I.) and by University of Texas Health School of Dentistry faculty funds to J.I. Deposited in PMC for release after 12 months.

The peer review history is available online at https://journals.biologists.com/dev/article-lookup/doi/10.1242/dev.200476.

Barritt
,
L. C.
,
Miller
,
J. M.
,
Scheetz
,
L. R.
,
Gardner
,
K.
,
Pierce
,
M. L.
,
Soukup
,
G. A.
and
Rocha-Sanchez
,
S. M.
(
2012
).
Conditional deletion of the human ortholog gene Dicer1 in Pax2-Cre expression domain impairs orofacial development
.
Indian J. Hum. Genet.
18
,
310
-
319
.
Beaty
,
T. H.
,
Marazita
,
M. L.
and
Leslie
,
E. J.
(
2016
).
Genetic factors influencing risk to orofacial clefts: today's challenges and tomorrow's opportunities
.
F1000Res.
5
,
2800
.
Bosman
,
E. A.
,
Penn
,
A. C.
,
Ambrose
,
J. C.
,
Kettleborough
,
R.
,
Stemple
,
D. L.
and
Steel
,
K. P.
(
2005
).
Multiple mutations in mouse Chd7 provide models for CHARGE syndrome
.
Hum. Mol. Genet.
14
,
3463
-
3476
.
Cao
,
H.
,
Yu
,
W.
,
Li
,
X.
,
Wang
,
J.
,
Gao
,
S.
,
Holton
,
N. E.
,
Eliason
,
S.
,
Sharp
,
T.
and
Amendt
,
B. A.
(
2016
).
A new plasmid-based microRNA inhibitor system that inhibits microRNA families in transgenic mice and cells: a potential new therapeutic reagent
.
Gene Ther.
23
,
527
-
542
.
Collins
,
M. D.
,
Eckhoff
,
C.
,
Weiss
,
R.
,
Resnick
,
E.
,
Nau
,
H.
and
Scott
,
W. J.
Jr.
(
2006
).
Differential teratogenesis of all-trans-retinoic acid administered on gestational day 9.5 to SWV and C57BL/6N mice: emphasis on limb dysmorphology
.
Birth Defects Res. A Clin. Mol. Teratol.
76
,
96
-
106
.
Cox
,
G. A.
,
Mahaffey
,
C. L.
,
Nystuen
,
A.
,
Letts
,
V. A.
and
Frankel
,
W. N.
(
2000
).
The mouse fidgetin gene defines a new role for AAA family proteins in mammalian development
.
Nat. Genet.
26
,
198
-
202
.
de Pontual
,
L.
,
Yao
,
E.
,
Callier
,
P.
,
Faivre
,
L.
,
Drouin
,
V.
,
Cariou
,
S.
,
Van Haeringen
,
A.
,
Geneviève
,
D.
,
Goldenberg
,
A.
,
Oufadem
,
M.
et al. 
(
2011
).
Germline deletion of the miR-17 approximately 92 cluster causes skeletal and growth defects in humans
.
Nat. Genet.
43
,
1026
-
1030
.
Dixon
,
M. J.
,
Marazita
,
M. L.
,
Beaty
,
T. H.
and
Murray
,
J. C.
(
2011
).
Cleft lip and palate: understanding genetic and environmental influences
.
Nat. Rev. Genet.
12
,
167
-
178
.
Dudas
,
M.
,
Kim
,
J.
,
Li
,
W. Y.
,
Nagy
,
A.
,
Larsson
,
J.
,
Karlsson
,
S.
,
Chai
,
Y.
and
Kaartinen
,
V.
(
2006
).
Epithelial and ectomesenchymal role of the type I TGF-β receptor ALK5 during facial morphogenesis and palatal fusion
.
Dev. Biol.
296
,
298
-
314
.
Eberhart
,
J. K.
,
He
,
X.
,
Swartz
,
M. E.
,
Yan
,
Y.-L.
,
Song
,
H.
,
Boling
,
T. C.
,
Kunerth
,
A. K.
,
Walker
,
M. B.
,
Kimmel
,
C. B.
and
Postlethwait
,
J. H.
(
2008
).
MicroRNA Mirn140 modulates Pdgf signaling during palatogenesis
.
Nat. Genet.
40
,
290
-
298
.
Enomoto
,
H.
,
Nelson
,
C. M.
,
Somerville
,
R. P.
,
Mielke
,
K.
,
Dixon
,
L. J.
,
Powell
,
K.
and
Apte
,
S. S.
(
2010
).
Cooperation of two ADAMTS metalloproteases in closure of the mouse palate identifies a requirement for versican proteolysis in regulating palatal mesenchyme proliferation
.
Development
137
,
4029
-
4038
.
Gong
,
S.-G.
,
White
,
N. J.
and
Sakasegawa
,
A. Y.
(
2000
).
The Twirler mouse, a model for the study of cleft lip and palate
.
Arch. Oral Biol.
45
,
87
-
94
.
Group
,
I. W.
(
2011
).
Prevalence at birth of cleft lip with or without cleft palate: data from the international perinatal database of typical oral clefts (IPDTOC)
.
Cleft Palate Craniofac. J.
48
,
66
-
81
.
Hu
,
S.
,
Huang
,
M.
,
Nguyen
,
P. K.
,
Gong
,
Y.
,
Li
,
Z.
,
Jia
,
F.
,
Lan
,
F.
,
Liu
,
J.
,
Nag
,
D.
,
Robbins
,
R. C.
et al. 
(
2011
).
Novel microRNA prosurvival cocktail for improving engraftment and function of cardiac progenitor cell transplantation
.
Circulation
124
,
S27
-
S34
.
Huang
,
H. Y.
,
Lin
,
Y. C.
,
Li
,
J.
,
Huang
,
K. Y.
,
Shrestha
,
S.
,
Hong
,
H. C.
,
Tang
,
Y.
,
Chen
,
Y. G.
,
Jin
,
C. N.
,
Yu
,
Y.
et al. 
(
2020
).
miRTarBase 2020: updates to the experimentally validated microRNA-target interaction database
.
Nucleic Acids Res.
48
,
D148
-
D154
.
Jin
,
J.
,
Jia
,
Z.-H.
,
Luo
,
X.-H.
and
Zhai
,
H.-F.
(
2020
).
Long non-coding RNA HOXA11-AS accelerates the progression of keloid formation via miR-124-3p/TGFβR1 axis
.
Cell Cycle
19
,
218
-
232
.
Kanoria
,
S.
,
Rennie
,
W.
,
Liu
,
C.
,
Carmack
,
C. S.
,
Lu
,
J.
and
Ding
,
Y.
(
2016
).
STarMir tools for prediction of microRNA binding sites
.
Methods Mol. Biol.
1490
,
73
-
82
.
Li
,
L.
,
Meng
,
T.
,
Jia
,
Z.
,
Zhu
,
G.
and
Shi
,
B.
(
2010
).
Single nucleotide polymorphism associated with nonsyndromic cleft palate influences the processing of miR-140
.
Am. J. Med. Genet. A
152A
,
856
-
862
.
Li
,
L.
,
Zhu
,
G. Q.
,
Meng
,
T.
,
Shi
,
J. Y.
,
Wu
,
J.
,
Xu
,
X.
and
Shi
,
B.
(
2011
).
Biological and epidemiological evidence of interaction of infant genotypes at Rs7205289 and maternal passive smoking in cleft palate
.
Am. J. Med. Genet. A
155A
,
2940
-
2948
.
Li
,
J.
,
Zou
,
J.
,
Li
,
Q.
,
Chen
,
L.
,
Gao
,
Y.
,
Yan
,
H.
,
Zhou
,
B.
and
Li
,
J.
(
2016
).
Assessment of differentially expressed plasma microRNAs in nonsyndromic cleft palate and nonsyndromic cleft lip with cleft palate
.
Oncotarget
7
,
86266
-
86279
.
Li
,
A.
,
Jia
,
P.
,
Mallik
,
S.
,
Fei
,
R.
,
Yoshioka
,
H.
,
Suzuki
,
A.
,
Iwata
,
J.
and
Zhao
,
Z.
(
2020
).
Critical microRNAs and regulatory motifs in cleft palate identified by a conserved miRNA-TF-gene network approach in humans and mice
.
Brief. Bioinform.
21
,
1465
-
1478
.
Miroshnichenko
,
S.
and
Patutina
,
O.
(
2019
).
Enhanced inhibition of tumorigenesis using combinations of miRNA-targeted therapeutics
.
Front. Pharmacol.
10
,
488
.
Mukherjee
,
S.
,
Diaz Valencia
,
J. D.
,
Stewman
,
S.
,
Metz
,
J.
,
Monnier
,
S.
,
Rath
,
U.
,
Asenjo
,
A. B.
,
Charafeddine
,
R. A.
,
Sosa
,
H. J.
,
Ross
,
J. L.
et al. 
(
2012
).
Human Fidgetin is a microtubule severing the enzyme and minus-end depolymerase that regulates mitosis
.
Cell Cycle
11
,
2359
-
2366
.
Mukhopadhyay
,
P.
,
Smolenkova
,
I.
,
Warner
,
D.
,
Pisano
,
M. M.
and
Greene
,
R. M.
(
2019
).
Spatio-temporal expression and functional analysis of miR-206 in developing orofacial tissue
.
MicroRNA
8
,
43
-
60
.
Nie
,
X.
,
Wang
,
Q.
and
Jiao
,
K.
(
2011
).
Dicer activity in neural crest cells is essential for craniofacial organogenesis and pharyngeal arch artery morphogenesis
.
Mech. Dev.
128
,
200
-
207
.
Pan
,
Y.
,
Li
,
D.
,
Lou
,
S.
,
Zhang
,
C.
,
Du
,
Y.
,
Jiang
,
H.
,
Zhang
,
W.
,
Ma
,
L.
and
Wang
,
L.
(
2018
).
A functional polymorphism in the pre-miR-146a gene is associated with the risk of nonsyndromic orofacial cleft
.
Hum. Mutat.
39
,
742
-
750
.
Pozzo
,
E.
,
Giarratana
,
N.
,
Sassi
,
G.
,
Elmastas
,
M.
,
Killian
,
T.
,
Wang
,
C.-C.
,
Marini
,
V.
,
Ronzoni
,
F.
,
Yustein
,
J.
,
Uyttebroeck
,
A.
et al. 
(
2021
).
Upregulation of miR181a/miR212 improves myogenic commitment in murine fusion-negative rhabdomyosarcoma
.
Front. Physiol.
12
,
701354
.
Rattanasopha
,
S.
,
Tongkobpetch
,
S.
,
Srichomthong
,
C.
,
Siriwan
,
P.
,
Suphapeetiporn
,
K.
and
Shotelersuk
,
V.
(
2012
).
PDGFRa mutations in humans with isolated cleft palate
.
Eur. J. Hum. Genet.
20
,
1058
-
1062
.
Ries
,
R. J.
,
Yu
,
W.
,
Holton
,
N.
,
Cao
,
H.
and
Amendt
,
B. A.
(
2017
).
Inhibition of the miR-17-92 cluster separates stages of palatogenesis
.
J. Dent. Res.
96
,
1257
-
1264
.
Seelan
,
R. S.
,
Mukhopadhyay
,
P.
,
Warner
,
D. R.
,
Appana
,
S. N.
,
Brock
,
G. N.
,
Pisano
,
M. M.
and
Greene
,
R. M.
(
2014
).
Methylated microRNA genes of the developing murine palate
.
MicroRNA
3
,
160
-
173
.
Shao
,
S.
,
Hu
,
Q.
,
Wu
,
W.
,
Wang
,
M.
,
Huang
,
J.
,
Zhao
,
X.
,
Tang
,
G.
and
Liang
,
T.
(
2020
).
Tumor-triggered personalized microRNA cocktail therapy for hepatocellular carcinoma
.
Biomater. Sci.
8
,
6579
-
6591
.
Shimizu
,
H.
,
Lee
,
G. S.
,
Beedanagari
,
S. R.
and
Collins
,
M. D.
(
2007
).
Altered localization of gene expression in both ectoderm and mesoderm is associated with a murine strain difference in retinoic acid-induced forelimb ectrodactyly
.
Birth Defects Res. A Clin. Mol. Teratol.
79
,
465
-
482
.
Stussel
,
L. G.
,
Hollstein
,
R.
,
Laugsch
,
M.
,
Hochfeld
,
L. M.
,
Welzenbach
,
J.
,
Schroder
,
J.
,
Thieme
,
F.
,
Ishorst
,
N.
,
Romero
,
R. O.
,
Weinhold
,
L.
et al. 
(
2022
).
MiRNA-149 as a candidate for facial clefting and neural crest cell migration
.
J. Dent. Res.
101
,
323
-
330
.
Suzuki
,
A.
,
Abdallah
,
N.
,
Gajera
,
M.
,
Jun
,
G.
,
Jia
,
P.
,
Zhao
,
Z.
and
Iwata
,
J.
(
2018a
).
Genes and microRNAs associated with mouse cleft palate: a systematic review and bioinformatics analysis
.
Mech. Dev.
150
,
21
-
27
.
Suzuki
,
A.
,
Jun
,
G.
,
Abdallah
,
N.
,
Gajera
,
M.
and
Iwata
,
J.
(
2018b
).
Gene datasets associated with mouse cleft palate
.
Data Brief
18
,
655
-
673
.
Suzuki
,
A.
,
Yoshioka
,
H.
,
Summakia
,
D.
,
Desai
,
N. G.
,
Jun
,
G.
,
Jia
,
P.
,
Loose
,
D. S.
,
Ogata
,
K.
,
Gajera
,
M. V.
,
Zhao
,
Z.
et al. 
(
2019
).
MicroRNA-124-3p suppresses mouse lip mesenchymal cell proliferation through the regulation of genes associated with cleft lip in the mouse
.
BMC Genomics
20
,
852
.
Takagi
,
T.
,
Moribe
,
H.
,
Kondoh
,
H.
and
Higashi
,
Y.
(
1998
).
DeltaEF1, a zinc finger and homeodomain transcription factor, is required for skeleton patterning in multiple lineages
.
Development
125
,
21
-
31
.
Wang
,
J.
,
Bai
,
Y.
,
Li
,
H.
,
Greene
,
S. B.
,
Klysik
,
E.
,
Yu
,
W.
,
Schwartz
,
R. J.
,
Williams
,
T. J.
and
Martin
,
J. F.
(
2013
).
MicroRNA-17-92, a direct Ap-2α transcriptional target, modulates T-box factor activity in orofacial clefting
.
PLoS Genet.
9
,
e1003785
.
Wang
,
X.
,
Gu
,
M.
,
Ju
,
Y.
and
Zhou
,
J.
(
2020
).
PIK3C3 acts as a tumor suppressor in esophageal squamous cell carcinoma and was regulated by MiR-340-5p
.
Med. Sci. Monit.
26
,
e920642
.
Warner
,
D. R.
,
Mukhopadhyay
,
P.
,
Brock
,
G.
,
Webb
,
C. L.
,
Michele Pisano
,
M.
and
Greene
,
R. M.
(
2014
).
MicroRNA expression profiling of the developing murine upper lip
.
Dev. Growth Differ.
56
,
434
-
447
.
Wong
,
N.
and
Wang
,
X.
(
2015
).
miRDB: an online resource for microRNA target prediction and functional annotations
.
Nucleic Acids Res.
43
,
D146
-
D152
.
Wu
,
Z. S.
,
Wu
,
Q.
,
Wang
,
C.-Q.
,
Wang
,
X.-N.
,
Huang
,
J.
,
Zhao
,
J.-J.
,
Mao
,
S.-S.
,
Zhang
,
G.-H.
,
Xu
,
X.-C.
and
Zhang
,
N.
(
2011
).
miR-340 inhibition of breast cancer cell migration and invasion through targeting of oncoprotein c-Met
.
Cancer
117
,
2842
-
2852
.
Xu
,
M.
,
Ma
,
L.
,
Lou
,
S.
,
Du
,
Y.
,
Yin
,
X.
,
Zhang
,
C.
,
Fan
,
L.
,
Wang
,
H.
,
Wang
,
Z.
,
Zhang
,
W.
et al. 
(
2018
).
Genetic variants of microRNA processing genes and risk of non-syndromic orofacial clefts
.
Oral Dis.
24
,
422
-
428
.
Xu
,
Y.
,
Xie
,
B.
,
Shi
,
J.
,
Li
,
J.
,
Zhou
,
C.
,
Lu
,
W.
,
Xu
,
F.
and
He
,
F.
(
2021a
).
Distinct expression of miR-378 in nonsyndromic cleft lip and/or cleft palate: a cogitation of skewed sex ratio in prevalence
.
Cleft Palate Craniofac. J.
58
,
61
-
71
.
Xu
,
Y.
,
Yuan
,
D.
,
Fan
,
Z.
,
Wang
,
S.
and
Du
,
J.
(
2021b
).
Identification and profiles of microRNAs in different development stages of miniature pig secondary palate
.
Genomics
113
,
2634
-
2644
.
Yan
,
F.
,
Jia
,
P.
,
Yoshioka
,
H.
,
Suzuki
,
A.
,
Iwata
,
J.
and
Zhao
,
Z.
(
2020
).
A developmental stage-specific network approach for studying dynamic co-regulation of transcription factors and microRNAs during craniofacial development
.
Development
147
,
dev192948
.
Yang
,
Y.
,
Mahaffey
,
C. L.
,
Bérubé
,
N.
and
Frankel
,
W. N.
(
2006
).
Interaction between fidgetin and protein kinase A-anchoring protein AKAP95 is critical for palatogenesis in the mouse
.
J. Biol. Chem.
281
,
22352
-
22359
.
Yoshioka
,
H.
,
Mikami
,
Y.
,
Ramakrishnan
,
S. S.
,
Suzuki
,
A.
and
Iwata
,
J.
(
2021a
).
MicroRNA-124-3p plays a crucial role in cleft palate induced by retinoic acid
.
Front. Cell Dev. Biol.
9
,
621045
.
Yoshioka
,
H.
,
Ramakrishnan
,
S. S.
,
Suzuki
,
A.
and
Iwata
,
J.
(
2021b
).
Phenytoin inhibits cell proliferation through microRNA-196a-5p in mouse lip mesenchymal cells
.
Int. J. Mol. Sci.
22
,
1746
.
Zehir
,
A.
,
Hua
,
L. L.
,
Maska
,
E. L.
,
Morikawa
,
Y.
and
Cserjesi
,
P.
(
2010
).
Dicer is required for survival of differentiating neural crest cells
.
Dev. Biol.
340
,
459
-
467
.
Zhao
,
P.
,
Ma
,
W.
,
Hu
,
Z.
,
Zhang
,
Y.
,
Zhang
,
S.
and
Wang
,
Y.
(
2018
).
Up-regulation of miR-340-5p promotes progression of thyroid cancer by inhibiting BMP4
.
J. Endocrinol. Investig.
41
,
1165
-
1172
.

Competing interests

The authors declare no competing or financial interests.

Supplementary information