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The role that noncoding regions of the genome play in the etiology of cleft palate is not well studied. A novel method of microRNA (miR) inhibition that allows for specific miR knockdown in vivo has been developed by our laboratory. To further understand the role of miRs in palatogenesis, we used a new mouse model to inhibit specific miRs within the miR-17-92 cluster. Transgenic mice expressing inhibitory complexes for miR-17 and miR-18 manifested a clefting phenotype that was distinct from that observed in mice carrying inhibitory complexes for miR-17, miR-18, miR-19, and miR-92. An in silico candidate gene analysis and bioinformatics review led us to identify TGFBR2 as a likely target of miR-17 and miR-19 family members. Reverse transcription polymerase chain reaction (RT-PCR) experiments showed that TGFBR1 and TGFBR2 expression levels were elevated in the palates of these miR transgenic embryos at embryonic day 15.5. RT-PCR data also showed that the expression of mature miRs from the miR-17-92 cluster was significantly decreased in the transgenic embryos. Decreased expression of TGFB pathway signaling ligands was also observed. Experiments in cells showed that inhibition of miR-17 and miR-18 was sufficient to induce increases in expression of TGFB receptors, while a concomitant decrease in TGFB signaling ligands was not observed. RT-PCR of mature miR-17-92 in cells demonstrated the selectivity and specificity of inhibitory complexes. While this study builds on previous studies that have implicated miR-17-92 in the regulation of important molecular components of the TGFB signaling pathway, it is likely that interactions remain to be elucidated between miR-17-92 and as-of-yet unidentified molecules important for the control of palatogenesis. The differential regulation of palatogenesis by members of the miR-17-92 cluster indicates that several gene combinations regulate palate elevation and extension during development.

The Iroquois genes (Irx) appear to regulate fundamental processes that lead to cell proliferation, differentiation, and maturation during development. In this report, the Iroquois homeobox 1 (Irx1) transcription factor was functionally disrupted using a LacZ insert and LacZ expression demonstrated stage-specific expression during embryogenesis. Irx1 is highly expressed in the brain, lung, digits, kidney, testis and developing teeth. Irx1 null mice are neonatal lethal and this lethality it due to pulmonary immaturity. Irx1−/− mice show delayed lung maturation characterized by defective surfactant protein secretion and Irx1 marks a population of SP-C expressing alveolar type II cells. Irx1 is specifically expressed in the outer enamel epithelium (OEE), stellate reticulum (SR) and stratum intermedium (SI) layers of the developing tooth. Irx1 mediates dental epithelial cell differentiation in the lower incisors resulting in delayed growth of the lower incisors. Irx1 is specifically and temporally expressed during developmental stages and we have focused on lung and dental development in this report. Irx1+ cells are unique to the development of the incisor outer enamel epithelium, patterning of Lef-1+ and Sox2+ cells as well as a new marker for lung alveolar type II cells. Mechanistically, Irx1 regulates Foxj1 and Sox9 to control cell differentiation during development.

Stress granules are biomolecular condensates composed of protein and mRNA. Long mRNAs are enriched in stress granules, which is thought to reflect the ability of long mRNAs to form multiple RNA-RNA interactions with other mRNAs. RNA-RNA interactions are thus thought to be critical for stress granule formation. Stress granule-enriched mRNAs also often contain multiple N6-methyladenosine (m6A) residues. YTHDF proteins bind m6A, creating mRNA-protein complexes that partition into stress granules. Here we determine the basis of length-dependent enrichment of mRNAs in stress granules. We show that depletion of m6A is sufficient to abrogate the length-dependent enrichment of mRNAs in stress granules. We show that the presence of m6A predicts which mRNAs are enriched. m6A formation is triggered by long exons, which are often found in long mRNAs, accounting for the link between m6A, length and stress granule enrichment. Thus, length-dependent enrichment of mRNAs in stress granules is driven by YTHDF-mRNA interactions.

Anaplastic thyroid cancer (ATC) is an aggressive, highly metastatic cancer that expresses high levels of the microRNA (miR)-17-92 cluster. We employ an miR inhibitor system to study the function of the different miRs within the miR-17-92 cluster based on seed sequence homology in the ATC SW579 cell line. While three of the four miR-17-92 families were oncogenic, we uncovered a novel role for miR-17 as a tumor suppressor in vitro and in vivo. Surprisingly, miR-17 inhibition increased expression of the miR-17-92 cluster and significantly increased the levels of the miR-18a and miR-19a mature miRs. miR-17 inhibition increased expression of the cell cycle activator CCND2, associated with increased cell proliferation and tumor growth in transplanted SW579 cells in xenograft mice. miR-17 regulates MYCN and c-MYC expression in SW579 cells, and the inhibition of miR-17 increased MYCN and c-MYC expression, which increased pri-miR-17-92 transcripts. Thus, inhibition of miR-17 activated the expression of the oncogenic miRs, miR-18a and miR-19a. While many cancers express high levels of miR-17, linking it with tumorigenesis, we demonstrate that miR-17 inhibition does not inhibit thyroid tumor growth in SW579 and MDA-T32 ATC cells but increases expression of the other miR-17-92 family members and genes to induce cancer progression.

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MicroRNAs (miRs) are small non‐coding RNAs that bind to target mRNAs and negatively regulate their expression. The expression of miRs are altered in many cancers and cancer cell types. We previously generated a miR‐17‐92 knockdown mouse model using the Plasmid based MicroRNA Inhibitor System (PMIS). With this new PMIS system we found that thyroid development was abnormal or absence in these mice. Thus, thyroid development requires miR‐17 for normal development. We investigated the role of miR‐17 in SW579 thyroid cancer cells, which are derived from human thyroid tissue. PMIS‐miR‐17, specifically targeting miR‐17‐5p completely inhibited the activity of miR‐17‐5p in SW579 cells. However, inhibiting miR‐17‐5p activity in SW579 thyroid cancer cells also promotes tumor growth in a nude mouse model for thyroid cancer. The tumor formation was due to increase cell proliferation and aberrant gene expression. RNA‐seq data shows knocking down miR‐17 in thyroid cancer cells induces large‐scale changes in miR‐17‐5p target gene expression, including PDGFR and MMP2, which may play oncogenic roles in tumorigenesis. Bioinformatics analyses has identified several new gene expression networks regulated by miR‐17 in thyroid cancer. We have identified several new gene expression mechanisms for miR‐17 and more importantly regulation of miR‐17‐92 expression and processing of the miR‐17‐92 cluster during thyroid development and thyroid cancer.Support or Funding InformationIowa Institute for Oral Health Research, College of Dentistry, The University of Iowa, Iowa City, IA