MicroRNA-124-3p suppresses mouse lip mesenchymal cell proliferation through the regulation of genes associated with cleft lip in the mouse

Background Cleft lip (CL), one of the most common congenital birth defects, shows considerable geographic and ethnic variation, with contribution of both genetic and environmental factors. Mouse genetic studies have identified several CL-associated genes. However, it remains elusive how these CL-associated genes are regulated and involved in CL. Environmental factors may regulate these genes at the post-transcriptional level through the regulation of non-coding microRNAs (miRNAs). In this study, we sought to identify miRNAs associated with CL in mice. Results Through a systematic literature review and a Mouse Genome Informatics (MGI) database search, we identified 55 genes that were associated with CL in mice. Subsequent bioinformatic analysis of these genes predicted that a total of 33 miRNAs target multiple CL-associated genes, with 20 CL-associated genes being potentially regulated by multiple miRNAs. To experimentally validate miRNA function in cell proliferation, we conducted cell proliferation/viability assays for the selected five candidate miRNAs (miR-124-3p, let-7a-5p, let-7b-5p, let-7c-5p, and let-7d-5p). Overexpression of miR-124-3p, but not of the others, inhibited cell proliferation through suppression of CL-associated genes in cultured mouse embryonic lip mesenchymal cells (MELM cells) isolated from the developing mouse lip region. By contrast, miR-124-3p knockdown had no effect on MELM cell proliferation. This miRNA-gene regulatory mechanism was mostly conserved in O9–1 cells, an established cranial neural crest cell line. Expression of miR-124-3p was low in the maxillary processes at E10.5, when lip mesenchymal cells proliferate, whereas it was greatly increased at later developmental stages, suggesting that miR-124-3p expression is suppressed during the proliferation phase in normal palate development. Conclusions Our findings indicate that upregulated miR-124-3p inhibits cell proliferation in cultured lip cells through suppression of CL-associated genes. These results will have a significant impact, not only on our knowledge about lip morphogenesis, but also on the development of clinical approaches for the diagnosis and prevention of CL.


Background
Cleft lip (CL) is one of the most common congenital birth defects, with a prevalence of 1/500 to 1/2500 live births worldwide. Approximately 70% of the cases of CL with/without cleft palate (CL/P) are non-syndromic (isolated CL/P), and the remaining 30% are syndromic, displaying many other clinical symptoms and features. The etiology of CL/P is very complex and multifactorial, resulting from the effect of genetic and environmental factors along with geographic, racial, and ethnic influences [1].
Mouse models are well established and have been extensively used to study the mechanisms of CL. Mouse lip formation is similar to that of humans, and the underlying molecular mechanism is well conserved in mice [2]. Mouse lip development begins at embryonic day (E) 10.0 of embryogenesis, when the surface ectoderm thickens bilaterally on the ventrolateral aspect of the frontonasal process to form the nasal placodes. The frontonasal process then expands around the nasal placodes, forming the nasal pits and the horseshoe-shaped medial and lateral nasal processes. The maxillary process then grows rapidly pushing the nasal pits medially, whereas the ventrolateral growth of the medial nasal process converts the round nasal pits into dorsally pointed slits at E10.5. At this stage, the medial nasal process and the maxillary process, with the lateral nasal process wedged in between them, comprise the upper lip, and the fusion of the lateral and medial nasal processes is initiated. By E11.0, the maxillary and medial nasal processes rapidly grow, pushing the lateral nasal process rostrally and fusing between the maxillary and medial nasal processes to form the upper lip [3]. Any failure in the development of the maxillary and nasal processes leads to CL [4].
Previous mouse genetic studies show that mutations in various genes are associated with orofacial cleft, which includes CL, cleft palate, and midfacial/midline cleft [5]. In addition, environmental factors can cause CL [6]. An increasing number of studies suggest that several CL genetic and epigenetic factors could be grouped according to their common functions (e.g. cell proliferation, differentiation) and pathways (e.g. growth factor signaling pathways). However, it remains elusive how CL-associated genes are regulated by epigenetic factors.
MicroRNAs play important role in the posttranscriptional regulation of protein-coding genes, and their altered expression may lead to various developmental defects and diseases [7,8]. In order to identify the molecular pathways essential for lip formation from the complex etiology of CL, we conducted a systematic review and mouse genome informatics (MGI) database search, followed by bioinformatic analyses, for both CLassociated genes and their related miRNAs. Candidate miRNAs were further tested experimentally in cell proliferation/survival assays and quantitative RT-PCR analyses of target CL-associated genes. This study will help extract molecular pathways and networks associated with CL from currently available data.

Study characteristics
In this study, we focused on CL; therefore, we included cleft lip only (CLO) and cleft lip and palate (CLP), but excluded midline cleft and cleft palate only (CPO). Our extensive literature search resulted in a total of 333 manuscripts. After screening the titles and abstracts of the articles, 152 studies were considered suitable for full-text review to identify the relevant articles; this initial screening was conducted by two screeners independently. As a result, we identified 45 eligible studies that were designed for the collection of causative genes for mouse CL ( Fig. 1 and Additional file 1). In these studies, a total of 25 genes [17 single gene mutants and six compound mutants (6 × 2 = 12 genes), with four duplicated genes excluded] and four spontaneous mouse lines with unknown mutation loci were validated as CL genes after the full-text review. In addition, we searched the MGI database, which stores collective information for mouse phenotypes, using the term "cleft lip"; 84 mouse lines were identified in this search. The 43 genes or alleles (51.2%) listed in the MGI database were not validated as CL genes because they were either a reporter gene, a Cre expression mouse line, had no CL phenotype, were a duplicate, or were excluded from the CL-associated gene list. As a result, a total of 41 genes [33 genes from single gene mutants and 8 genes from compound mutants after excluding six duplicated genes; 48.8%] were identified as CL-associated genes in the MGI database (Fig. 2).
The bibliographies of highly pertinent articles were further examined to avoid any errors introduced with the systematic review. As a result, we found a total of 55 genes as CL-associated genes. Among them, a total of 39 genes were identified in mice with CL/P resulting from a single gene deficiency (Table 1). There are nine spontaneous CL/P mouse lines (four genes after excluding any duplicated genes; five mouse lines with spontaneous mutations in CL-associated genes and four mouse lines with spontaneous mutations in unknown gene and loci). The penetrance of CL/P in spontaneous mouse lines is quite low (less than 40%) ( Table 2). Ten compound mutant mice (mice with two mutant genes; 12 genes after excluding any duplicated genes) exhibited CL (Table 3). Among these 55 CL-associated genes, 20.0% (11 out of 55 genes) were common in the systematic review and MGI database search. There were 14 genes (25.5%, 14 out of 55 genes) and 30 genes (54.5%, 30 out of 55 genes) uniquely identified through the systematic review and MGI search, respectively (Fig. 2).

Environmental and epigenetic factors
The prevalence of CL is influenced by genetic background, ethnicity, and gender. In addition, maternal conditions (e.g. age, smoking, alcohol consumption, obesity, micronutrient deficiencies) affect CL prevalence. MicroRNAs (miRNAs), short (~22 nucleotides) noncoding RNAs [67] that control gene expression at the post-transcriptional level [68], are known to be altered by maternal conditions and environmental factors. To identify miRNAs that can regulate the expression of CL genes, we carried out a miRNA-target gene enrichment analysis for CL-associated genes. With an adjusted p-value < 0.2, we identified 33 miRNAs whose target genes were significantly enriched with the CL genes (Table 4). Among them were miR-124-3p and let-7 family members (let-7a-1-3p, let-7b-3p, let-7c-2-3p, let-7f-1-3p), for which previous miRNA profiling indicated a spatiotemporal-specific expression in the medial nasal and maxillary processes during lip development [70]. These results suggest that miR-124-3p and let-7 family members may play crucial role in lip development. Among the miRNA targets, Zeb1 was the most frequently targeted gene, by 17 out of 33 miRNAs, followed by Pbx1, Pbx3, Ptch1, and Sox11, targeted by 16 miRNAs (Table 5). These results suggest that miRNAs may play a crucial role in the pathology of CL through the regulation of CLassociated genes.    Experimental validation miRNAs suppress multiple target mRNAs [71]. Because loss of function of CL-associated genes causes CL in mice, we tested whether overexpression of these miR-NAs inhibited cell proliferation through the suppression of target genes. To test this hypothesis, we used primary mouse embryonic upper lip mesenchymal (MELM) cells isolated from the developing upper lip region ( Fig. 3a), which were then treated with each miRNA mimic. The miR-124-3p mimic significantly inhibited cell proliferation in MELM cells isolated from the developing lip regions; by contrast, treatment with mimics for let-7a-5p, let-7b-5p, let-7c-5p, and let-7d-5p resulted in no proliferation defect (Fig. 3b, c). We also confirmed that the miR-124-3p mimic did not induce apoptosis (Fig. 3d).
To identify target genes regulated by miR-124-3p, we performed quantitative RT-PCR analyses for the predicted target genes in MELM cells after treatment with the miR-124-3p mimic and observed that expression of Bmpr1a, Cdc42, Ift88, Pbx3 and Tgfbr1 was significantly downregulated (Fig. 4). Next, to examine the effect of loss-of-function of miR-124-3p in cell proliferation and CL-associated gene regulation, we performed cell proliferation assays and quantitative RT-PCR analyses for CL-associated genes in cells treated with a miR-124-3p inhibitor. We found that miR-124-3p inhibition did not affect cell proliferation in MELM cells isolated from either E10.5 or E11.5 maxillary processes (Fig. 5a, c). This indicates that loss-of-function of miR-124-3p has less impact on cell proliferation during lip development. Cdc42 and Pbx3, which were suppressed by miR-124-3p overexpression, were upregulated upon treatment with miR-124-3p inhibitor in MELM cells (Fig. 5b, d),  suggesting that the expression of these genes is regulated by miR-124-3p in a dose-dependent manner and that they may be accurate target genes of miR-124-3p in lip development. Next, we examined when and where miR-124-3p was expressed during normal lip development. Expression of miR-124-3p was slightly upregulated at E12.5, and greatly increased at E13.5, in the maxillary process during lip development (Fig. 6a). The expression of the predicted target genes was anti-correlated with miR-124-3p expression in the maxillary process at E10.5 to E13.5 (Fig. 6b).
To examine the conservation of these phenotypes in other cell types that are similar to mouse lip cells, we analyzed O9-1 cells, an established cranial neural crest cell line isolated from E8.5 mouse embryos, after treatment with a miR-124-3p mimic. As expected, miR-124-3p strongly suppressed cell proliferation (Fig. 7a). By contrast, the miR-124-3p inhibitor did not alter O9-1 cell proliferation (Fig. 7b), as seen for MELM cells. Next, the expression of the predicted target genes was examined in O9-1 cells in order to compare it with that of MELM cells. We found that expression of Bmpr1a, Cdc42, Pbx3, and Tgfbr1 was suppressed by the miR-124-3p mimic, as seen in MELM cells (Fig. 6, c, d). In addition, during nasal process development, miR-124-3p overexpression inhibited cell proliferation in primary cells isolated from E11.5 medial nasal processes, as seen for MELM cells. Furthermore, the expression of miR-124-3p and its target genes was similarly changed during nasal process development (Additional file 2).
Taken together, our results indicate that upregulated miR-124-3p results in suppressed cell proliferation through CL-associated gene expression in cultured MELM and O9-1 cells.

Discussion
CL with or without cleft palate is part of the clinical features of approximately 400 known human syndromes [5]. A significant number of genetic mutations have been  Recently, a growing number of miRNA profiling studies clarified the contribution of miRNAs to nonsyndromic CL/P [72][73][74]. The contribution of miRNAs to CL has been elucidated using mice with a deletion of Dicer, a crucial enzyme for miRNA maturation [75]. Mice with the Dicer deletion in cranial neural crest (CNC) cells and lip mesenchymal cells exhibit severe craniofacial anomalies, including CL, through decreased cell proliferation and increased cell death [76,77], indicating that mesenchymal miRNAs play essential roles in lip development. By contrast, mice with the Dicer deletion in the lip epithelium (Dicer F/F ;K14-Cre or Dicer F/F ; Shh-Cre mice: K14-Cre and Shh-Cre are specifically expressed in the differentiating epithelium) exhibit no CL or craniofacial deformities [78,79]. This suggests that miRNAs may be less important in the lip epithelium compared to the mesenchyme. However, recent studies indicate that a Dicer-independent pathway exists in the miRNA maturation process [80]. Because the contribution of Dicer-independent miRNAs to lip fusion remains unknown, future genetic studies will identify the role of Dicer-independent miRNAs during lip formation.
In our experimental validation, we validated that miR-124-3p suppresses cell proliferation in cultured mouse lip mesenchymal cells. In nasopharyngeal carcinoma cells, miR-124-3p inhibits cell growth and metastasis formation by targeting STAT3 [81]. By contrast, let-7a-d failed to suppress cell proliferation in cultured lip mesenchymal cells, while let-7a inhibits cell proliferation in gastric cancer cells [82]. Although other miRNAs would potentially regulate the expression of these genes, our miRNA predictions did not reach significance for any other miRNAs. In cases when we did not see a consistent and dose-dependent change with miR-124-3p, these genes' expression might undergo a more complex regulation by other miRNAs, a combination of miR-124-3p and other miRNAs, or they may be suppressed at the protein translation level. Our results also suggest that each miRNA functions in a cell-specific manner.

Conclusions
The results from this study are important to understand the mechanisms and etiology of CL, to further validate CL-associated genes and their regulation in CL, and to design future clinical applications to prevent and diagnose CL in humans. It has been known that expression of miRNAs is altered by extracellular conditions. Our results suggest that upregulated miR-124-3p may cause CL through the suppression of CL-associated genes. This new knowledge has potential relevance for the pathways and networks of CL-associated genes and miRNAs in the regulation of the development of the lip.

Information sources for the gene search
We followed a guideline set forth by PRISMA (Preferred Reporting Items for Systematic Reviews and The drawings on the right show a mouse head at each developmental stage. Color code: frontonasal process, green; maxillary process, red; nasal process, light blue; and mandibular process, gray. b, c Cell proliferation assays using MELM cells from E10.5 (B) and E11.5 (c) lips treated with the indicated miRNAs. Negative control (control, light blue), miR-124-3p (orange), let-7a-5p (gray), let-7b-5p (yellow), let-7c-5p (blue), and let-7d-5p (light green). ** p < 0.01, *** p < 0.001. s Immunoblotting analysis for cleaved caspase 3 in MELM cells treated with negative control (NC), miR-124-3p mimic, and positive control (PC). GAPDH was used as an internal control Meta-Analyses) [83] for the systematic CL gene search. Public online databases Medline (Ovid), Embase (Ovid), and PubMed (NLM) were searched for articles and information on mouse CL-associated genes. In order to recover any missing data related to CL, we searched Scopus (Elsevier) and the MGI database. RefWorks was used for sorting the references and excluding duplicates from the systematic review, as described previously [84].

Eligibility criteria for the systematic review
The following inclusion criteria were applied in the selection of the articles: 1) genetic studies for mouse CL; 2) original articles (no review articles, editorials, or comments); 3) published in English; 4) articles specifying the genes responsible for CL in mice.
After the step above, we manually excluded those studies meeting one or more of the following criteria: 1) conducted primarily in other species; 2) describing environmental factors for CL instead of genetic factors.

Search strategy to identify the studies
A systematic literature search was conducted independently by two screeners using the Medline (Ovid), PubMed (NLM), and Embase (Ovid) databases. To conduct the search, Medical Subject Headings (MeSH) terms were developed, as described previously [85]. Different combinations and variations of the term 'CL' (i.e. CL, CL/P, CL and palate) were searched along with other terms such as 'mice' (or 'mouse'), 'genetics', and 'mutation'. Additionally, the bibliographies of the relevant articles were manually examined in Scopus (Elsevier) to retrieve studies that were not identified in the database searches.

Study design and case selection
RefWorks (ProQuest) and systematic review Excel workbooks were used to store and track all citations found in the search process and to eliminate duplicates. The Kappa statistic was used to determine the level of agreement between the two screeners. Full-text articles for which there was a disagreement were re-evaluated based on the inclusion criteria. A codebook for data extraction from the articles meeting the eligibility criteria was developed as previously described [84].

Bioinformatic analysis
The miRNA-target gene relationships were collected from four resources, including miRTarbase, a database of experimentally validated miRNA-gene interactions [86], and three databases for predicted miRNA-gene interactions (miRanda [87], PITA [88] and TargetScan [89]). The Fisher's exact test was used to test the in the maxillary process (MxP) from E10.5 to E13.5. * p < 0.05, ** p < 0.01, *** p < 0.001 significance level of the shared genes between miRNA target genes and mouse CL-associated genes. The Benjamini-Hochberg method was used for multiple test correction [69].

Animals
C57BL6/J mice were obtained from The Jackson Laboratory. All mice were maintained in the animal facility of UTHealth. The protocol was reviewed and approved by the Animal Welfare Committee (AWC) and the Institutional Animal Care and Use Committee (IACUC) of UTHealth.

Immunoblotting
Immunoblots were performed as described previously [91], using a rabbit polyclonal antibody against cleaved caspase 3 (Cell Signaling Technology) and a mouse monoclonal antibody against GAPDH (MilliporeSigma).

Statistical analysis
A two-tailed Student's t test was applied for the statistical analysis. A p value < 0.05 was considered statistically significant. For all graphs, data were parametric and represented as mean ± standard deviation (SD).
Additional file 1: The information of the databases searched.