{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,5]],"date-time":"2026-05-05T11:39:13Z","timestamp":1777981153948,"version":"3.51.4"},"reference-count":28,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,2,24]],"date-time":"2026-02-24T00:00:00Z","timestamp":1771891200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Interethnos-Interplast Italy"},{"DOI":"10.13039\/501100003770","name":"Banca d\u2019Italia","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100003770","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100016990","name":"BENEFICENTIA Stiftung","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100016990","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Cleft palate only (CPO) is a multifactorial craniofacial malformation with significant genetic and epigenetic contributions. Among these, microRNAs (miRNAs) have emerged as key regulators of palate development, although their alterations in CPO remain incompletely characterized. In this study, we performed a comprehensive miRNA expression analysis on palatal tissues from an Italian cohort of non-syndromic CPO patients, compared with a human embryonic palatal mesenchymal (HEPM) cell line. Using the NanoString\u00ae nCounter\u00ae platform for miRNA profiling, we identified significant deregulation of several miRNAs, notably the upregulation of miR-205-5p and miR-200c-3p and the downregulation of miR-125a-5p in CPO tissues. Based on these expression changes, a functional analysis was carried out to identify potential target genes. Validation in primary cell cultures derived from patient tissues confirmed these expression patterns. Functional analyses and target predictions implicated PAX9, a key transcription factor essential for palatogenesis, as a probable target of miR-205-5p, while miR-125a-5p was associated with the regulation of PRTG and PRSS35\u2014genes involved in neural crest cell biology and extracellular matrix remodeling, respectively. Although modulation of certain predicted targets of miR-200c-3p was observed, in vitro inhibition experiments did not show significant changes in gene expression, highlighting the complexity of miRNA regulatory networks and the need for further studies to unravel these interactions. These findings identify miRNA alterations associated with CPO tissue and fibroblasts, highlighting novel candidate pathways for further mechanistic and therapeutic investigation.<\/jats:p>","DOI":"10.3390\/ijms27052088","type":"journal-article","created":{"date-parts":[[2026,2,24]],"date-time":"2026-02-24T08:54:56Z","timestamp":1771923296000},"page":"2088","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Toward Understanding the Role of miRNAs in Cleft Palate Only: Observations from Patient Tissues and In Vitro Assays"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6338-7394","authenticated-orcid":false,"given":"Annalisa","family":"Palmieri","sequence":"first","affiliation":[{"name":"Department of Medical and Surgical Sciences, University of Bologna, 40126 Bologna, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4006-9910","authenticated-orcid":false,"given":"Luca","family":"Scapoli","sequence":"additional","affiliation":[{"name":"Department of Medical and Surgical Sciences, University of Bologna, 40126 Bologna, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Agnese","family":"Pellati","sequence":"additional","affiliation":[{"name":"Department of Translational Medicine and for Romagna, University of Ferrara, 44121 Ferrara, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1991-4115","authenticated-orcid":false,"given":"Federico","family":"Apolloni","sequence":"additional","affiliation":[{"name":"Regional Center for Diagnosis and Treatment of Cranio-Maxillofacial Malformation, Maxillofacial Unit, Ospedale San Bortolo, 36100 Vicenza, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-5697-8599","authenticated-orcid":false,"given":"Valerio","family":"Zanchi","sequence":"additional","affiliation":[{"name":"Department of Neurosurgery, Meyer Children\u2019s Hospital IRCCS, 50139 Florence, Italy"},{"name":"Department of Otorhinolaringology, ASST Valtellina e Alto Lario, 23100 Sondrio, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3834-0433","authenticated-orcid":false,"given":"Giuseppe","family":"Spinelli","sequence":"additional","affiliation":[{"name":"Maxillo-Facial Unit, Azienda Ospedaliero Universitaria Careggi, 50134 Florence, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5543-8013","authenticated-orcid":false,"given":"Rossella","family":"Sgarzani","sequence":"additional","affiliation":[{"name":"Department of Medical and Surgical Sciences, University of Bologna, 40126 Bologna, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9639-6676","authenticated-orcid":false,"given":"Francesco","family":"Carinci","sequence":"additional","affiliation":[{"name":"Department of Translational Medicine and for Romagna, University of Ferrara, 44121 Ferrara, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0219-7890","authenticated-orcid":false,"given":"Marcella","family":"Martinelli","sequence":"additional","affiliation":[{"name":"Department of Medical and Surgical Sciences, University of Bologna, 40126 Bologna, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,2,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"126","DOI":"10.1002\/(SICI)1096-8628(19980113)75:2<126::AID-AJMG2>3.0.CO;2-R","article-title":"Classification and birth prevalence of orofacial clefts","volume":"75","author":"Tolarova","year":"1998","journal-title":"Am. J. Med. Genet."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"410","DOI":"10.1016\/j.tig.2024.02.004","article-title":"The heterogeneous genetic architectures of orofacial clefts","volume":"40","author":"Robinson","year":"2024","journal-title":"Trends Genet."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Martinelli, M., Palmieri, A., Carinci, F., and Scapoli, L. (2020). Non-syndromic Cleft Palate: An Overview on Human Genetic and Environmental Risk Factors. Front. Cell Dev. Biol., 8.","DOI":"10.3389\/fcell.2020.592271"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"744","DOI":"10.1016\/j.ajhg.2016.02.014","article-title":"A Genome-wide Association Study of Nonsyndromic Cleft Palate Identifies an Etiologic Missense Variant in GRHL3","volume":"98","author":"Leslie","year":"2016","journal-title":"Am. J. Hum. Genet."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.diff.2022.01.002","article-title":"MicroRNAs as epigenetic regulators of orofacial development","volume":"124","author":"Seelan","year":"2022","journal-title":"Differentiation"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e05005","DOI":"10.7554\/eLife.05005","article-title":"Predicting effective microRNA target sites in mammalian mRNAs","volume":"4","author":"Agarwal","year":"2015","journal-title":"eLife"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1016\/j.ydbio.2023.10.007","article-title":"Exploring the roles of noncoding RNAs in craniofacial abnormalities: A systematic review","volume":"505","author":"Shi","year":"2024","journal-title":"Dev. Biol."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Im, H., Song, Y., Kim, J.K., Park, D.K., Kim, D.S., Kim, H., and Shin, J.O. (2025). Molecular Regulation of Palatogenesis and Clefting: An Integrative Analysis of Genetic, Epigenetic Networks, and Environmental Interactions. Int. J. Mol. Sci., 26.","DOI":"10.20944\/preprints202501.0962.v1"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Burg, M.L., Chai, Y., Yao, C.A., Magee, W., and Figueiredo, J.C. (2016). Epidemiology, Etiology, and Treatment of Isolated Cleft Palate. Front. Physiol., 7.","DOI":"10.3389\/fphys.2016.00067"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Suzuki, A., Li, A., Gajera, M., Abdallah, N., Zhang, M., Zhao, Z., and Iwata, J. (2019). MicroRNA-374a, -4680, and -133b suppress cell proliferation through the regulation of genes associated with human cleft palate in cultured human palate cells. BMC Med. Genom., 12.","DOI":"10.1186\/s12920-019-0546-z"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2570","DOI":"10.3892\/etm.2017.4248","article-title":"A pilot study: Screening target miRNAs in tissue of nonsyndromic cleft lip with or without cleft palate","volume":"13","author":"Wang","year":"2017","journal-title":"Exp. Ther. Med."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1016\/bs.ctdb.2015.07.002","article-title":"Cellular and Molecular Mechanisms of Palatogenesis","volume":"115","author":"Lan","year":"2015","journal-title":"Curr. Top. Dev. Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"3819","DOI":"10.1242\/dev.157750","article-title":"Small-molecule Wnt agonists correct cleft palates in Pax9 mutant mice in utero","volume":"144","author":"Jia","year":"2017","journal-title":"Development"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1273","DOI":"10.1177\/0022034517719865","article-title":"Modulating Wnt Signaling Rescues Palate Morphogenesis in Pax9 Mutant Mice","volume":"96","author":"Li","year":"2017","journal-title":"J. Dent. Res."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Almaidhan, A., Cesario, J., Landin Malt, A., Zhao, Y., Sharma, N., Choi, V., and Jeong, J. (2014). Neural crest-specific deletion of Ldb1 leads to cleft secondary palate with impaired palatal shelf elevation. BMC Dev. Biol., 14.","DOI":"10.1186\/1471-213X-14-3"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/j.bbrc.2023.04.090","article-title":"Increased miR-200c levels disrupt palatal fusion by affecting apoptosis, cell proliferation, and cell migration","volume":"664","author":"Won","year":"2023","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"8155","DOI":"10.1007\/s11033-021-06784-1","article-title":"Nuclear receptor subfamily 5 group A member 2 (NR5A2): Role in health and diseases","volume":"48","author":"Sandhu","year":"2021","journal-title":"Mol. Biol. Rep."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"4810","DOI":"10.1182\/blood-2008-07-170316","article-title":"Distinctive localization and opposed roles of vasohibin-1 and vasohibin-2 in the regulation of angiogenesis","volume":"113","author":"Kimura","year":"2009","journal-title":"Blood"},{"key":"ref_19","first-page":"469","article-title":"Evidence for gene-environment interaction in a genome wide study of nonsyndromic cleft palate","volume":"35","author":"Beaty","year":"2011","journal-title":"Genet. Epidemiol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"e651","DOI":"10.1038\/cddis.2013.177","article-title":"Protogenin prevents premature apoptosis of rostral cephalic neural crest cells by activating the alpha5beta1-integrin","volume":"4","author":"Wang","year":"2013","journal-title":"Cell Death Dis."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1531","DOI":"10.1002\/ajmg.a.38210","article-title":"Association studies of low-frequency coding variants in nonsyndromic cleft lip with or without cleft palate","volume":"173","author":"Leslie","year":"2017","journal-title":"Am. J. Med. Genet. A"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"26492","DOI":"10.1038\/s41598-024-77724-9","article-title":"Combining genetic and single-cell expression data reveals cell types and novel candidate genes for orofacial clefting","volume":"14","author":"Siewert","year":"2024","journal-title":"Sci. Rep."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"927","DOI":"10.1177\/0022034510370004","article-title":"Novel cleft susceptibility genes in chromosome 6q","volume":"89","author":"Letra","year":"2010","journal-title":"J. Dent. Res."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1016\/j.jcms.2015.07.026","article-title":"A multicentric association study between 39 genes and nonsyndromic cleft lip and palate in a Brazilian population","volume":"44","author":"Secolin","year":"2016","journal-title":"J. Craniomaxillofac. Surg."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Balcells, I., Cirera, S., and Busk, P.K. (2011). Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers. BMC Biotechnol., 11.","DOI":"10.1186\/1472-6750-11-70"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Busk, P.K. (2014). A tool for design of primers for microRNA-specific quantitative RT-qPCR. BMC Bioinform., 15.","DOI":"10.1186\/1471-2105-15-29"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S., and Madden, T.L. (2012). Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinform., 13.","DOI":"10.1186\/1471-2105-13-134"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1006\/meth.2001.1262","article-title":"Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method","volume":"25","author":"Livak","year":"2001","journal-title":"Methods"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/5\/2088\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,26]],"date-time":"2026-02-26T05:19:02Z","timestamp":1772083142000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/5\/2088"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,2,24]]},"references-count":28,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2026,3]]}},"alternative-id":["ijms27052088"],"URL":"https:\/\/doi.org\/10.3390\/ijms27052088","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,2,24]]}}}