MicroRNA-374a, -4680, and -133b suppress cell proliferation through the regulation of genes associated with human cleft palate in cultured human palate cells

Abstract

Background Cleft palate (CP) is the second most common congenital birth defect; however, the relationship between CP-associated genes and epigenetic regulation remains largely unknown. In this study, we investigated the contribution of microRNAs (miRNAs) to cell proliferation and regulation of genes involved in CP development. Methods In order to identify all genes for which mutations or association/linkage have been found in individuals with CP, we conducted a systematic literature search, followed by bioinformatics analyses for these genes. We validated the bioinformatics results experimentally by conducting cell proliferation assays and miRNA-gene regulatory analyses in cultured human palatal mesenchymal cells treated with each miRNA mimic. Results We identified 131 CP-associated genes in the systematic review. The bioinformatics analysis indicated that the CP genes were associated with signaling pathways, microRNAs (miRNAs), metabolic pathways, and cell proliferation. A total 17 miRNAs were recognized as potential modifiers of human CP genes. To validate miRNA function in cell proliferation, a main cause of CP, we conducted cell proliferation/viability assays for the top 11 candidate miRNAs from our bioinformatics analysis. Overexpression of miR-133b, miR-374a-5p, and miR-4680-3p resulted in a more than 30% reduction in cell proliferation activity in human palatal mesenchymal cell cultures. We found that several downstream target CP genes predicted by the bioinformatics analyses were significantly downregulated through induction of these miRNAs ( FGFR1 , GCH1 , PAX7 , SMC2 , and SUMO1 by miR-133b; ARNT , BMP2 , CRISPLD1 , FGFR2 , JARID2 , MSX1 , NOG , RHPN2 , RUNX2 , WNT5A and ZNF236 by miR - 374a-5p; and ERBB2 , JADE1 , MTHFD1 and WNT5A by miR-4680-3p) in cultured cells. Conclusions Our results indicate that miR-374a-5p, miR-4680-3p, and miR-133b regulate expression of genes that are involved in the etiology of human CP, providing insight into the association between CP-associated genes and potential targets of miRNAs in palate development. Electronic supplementary material The online version of this article (10.1186/s12920-019-0546-z) contains supplementary material, which is available to authorized users.

Role of epigenetics and miRNAs in orofacial clefts

“…Ding et al (2013) used the zebrafish larval model to identify miR‐133b as a miRNA whose overexpression resulted in midfacial cleft (Ding et al, 2016). These findings were corroborated by Suzuki et al (2019) using human PMCs (Suzuki, Li, et al, 2019). Among the validated target genes of miR‐133b in human cells were key regulators of palatogenesis, including an Fgf signaling receptor FGFR1 and the transcription factor PAX7 .…”

“…Two recent studies leveraged the literature and in silico modeling to identify miRNAs associated with OFCs and subsequently validate predicted target genes in cultured human cells. Suzuki et al (2019) identified miR‐133b, miR‐374a‐5p, and miR‐4680‐3p as being differentially regulated in CPO patients (Suzuki, Li, et al, 2019). The latter two miRNAs were validated to target WNT5A expression using miRNA mimics in human PMCs.…”

Gene–environment interplay and MicroRNAs in cleft lip and cleft palate

“…A total of 177 gene mutations has been reported in cases of CL/P in humans, 13,14 and a total of 252 genes has been identified in genetically mutated mouse models exhibiting CP 28 . Among them, 49 genes ( Alx1 , Alx3 , Bmp4 , Chd7 , Col2a1 , Col11a1 , Dhcr7 , Efnb1 , Eya1 , Fgf8 , Fgf10 , Fgfr1 , Fgfr2 , Flna, Foxc2 , Foxe1 , Gli2 , Gli3 , Grhl3 , Hic1 , Irf6 , Jag2 , Kcnj2 , Msx1 , Ofd1 , Pax9 , Pdgfc , Prickle1 , Ptch1 , Recql4 , Ror2 , Satb2 , Six3 , Ski , Sox9 , Sox11 , Sumo1 , Tbx1 , Tbx22 , Tcof1 , Tfap2a , Tgfb3, Tgfbr1 , Tgfbr2 , Trp63 , Twist1 , Vax1 , Wnt5a , and Zeb2 ) are common between humans and mice (Table 3).…”

“…[8][9][10][11] Approximately 70% of CL/P is non-syndromic, but more than 487 syndromes in which CL/P is a part of the clinical features have been reported. [12][13][14] Asian and Native American populations have the highest prevalence, as high as 1 in 500, and African populations have the lowest prevalence, at approximately 1 in 2500. The occurrence of CL/P also differs by gender and laterality.…”

“…Although a few factors have been suggested to be associated with orofacial cleft (eg, folic acid supplementation before and during pregnancy and mother's cigarette smoking and alcohol consumption during pregnancy), the exact mechanisms of these factors remain largely unknown 8‐11 . Approximately 70% of CL/P is non‐syndromic, but more than 487 syndromes in which CL/P is a part of the clinical features have been reported 12‐14 . Asian and Native American populations have the highest prevalence, as high as 1 in 500, and African populations have the lowest prevalence, at approximately 1 in 2500.…”