An miRNA derived from amelogenin exon4 regulates expression of transcription factor Runx2 by directly targeting upstream activators Nfia and Prkch.
An miRNA derived from amelogenin exon4 regulates expression of transcription factor Runx2 by directly targeting upstream activators Nfia and Prkch.
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DOI：
10.1016/j.jbc.2022.101807
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发表时间：
2022-05
影响因子：
4.8
通讯作者：
Nakano, Yukiko
中科院分区：
文献类型：
--
作者：
Shemirani, Rozana;Lin, Gan;Uyghurturk, Dawud Abduweli;Le, Michael;Nakano, Yukiko
Amel是编码淀粉原蛋白的基因，参与了牙釉质的形成，是高度选择性剪接的。当exon4被切除时，它可以形成一个成熟的miRNA (miR-exon4)，该miRNA先前被认为间接调节runt相关转录因子2 （Runx2）的表达，参与成釉细胞和成骨细胞的骨发育。然而，这种监管的确切机制尚不清楚。在这项研究中，我们旨在确定miR-exon4的直接靶点。已知转录因子家族核因子I/A （NFI/A）可以负向调节Runx2的表达，并且是与Runx2相关的miR-exon4最可预测的直接靶点之一。免疫染色在体内成骨细胞和成釉细胞中检测到NFI/A，报告基因检测证实了Nfia 3 ' -UTR和miR-exon4的直接相互作用。此外，Nfia在MC3T3-E1-M14成骨细胞中的沉默导致Runx2随后下调。在成熟的miR-exon4被功能抑制的MC3T3-E1细胞的单克隆亚克隆（mi2）中，我们观察到Runx2的表达显著下调。我们发现NFI/A在mi2细胞的mRNA和蛋白水平上均显著上调。此外，对mi2细胞中基因表达的定量蛋白质组学和通路分析表明，miR-exon4可以直接靶向Prkch（蛋白激酶C-eta），可能通过雷帕霉素激酶活化的机制靶点调控RUNX2。报告基因分析也证实了miR-exon4与Prkch的3 ' -UTR的直接相互作用，Western blot分析证实了mi2细胞中雷帕霉素激酶磷酸化的机制靶点显著上调。综上所述，我们得出结论，Nfia和Prkch的表达与miR-exon4介导的体内和体外Runx2调控负相关，表明miR-exon4直接靶向Nfia和Prkch调控Runx2。
Amel, the gene encoding the amelogenin protein involved in enamel formation, is highly alternatively spliced. When exon4 is excised, it can form a mature miRNA (miR-exon4) that has previously been suggested to indirectly regulate expression of the Runt-related transcription factor 2 (Runx2) involved in bone development in ameloblasts and osteoblasts. However, the precise mechanism of this regulation is unclear. In this study, we aimed to identify direct targets of miR-exon4. The transcription factor family nuclear factor I/A (NFI/A) is known to negatively regulate expression of Runx2 and is among the most highly predicted direct targets of miR-exon4 that link to Runx2. Immunostaining detected NFI/A in osteoblasts and ameloblasts in vivo, and reporter assays confirmed direct interaction of the Nfia 3′-UTR and miR-exon4. In addition, silencing of Nfia in MC3T3-E1-M14 osteoblasts resulted in subsequent downregulation of Runx2. In a monoclonal subclone (mi2) of MC3T3-E1 cells wherein mature miR-exon4 was functionally inhibited, we observed significantly downregulated Runx2 expression. We showed that NFI/A was significantly upregulated in mi2 cells at both mRNA and protein levels. Furthermore, quantitative proteomics and pathway analysis of gene expression in mi2 cells suggested that miR-exon4 could directly target Prkch (protein kinase C-eta), possibly leading to RUNX2 regulation through mechanistic target of rapamycin kinase activation. Reporter assays also confirmed the direct interaction of miR-exon4 and the 3′-UTR of Prkch, and Western blot analysis confirmed significantly upregulated mechanistic target of rapamycin kinase phosphorylation in mi2 cells. Taken together, we conclude that Nfia and Prkch expression negatively correlates with miR-exon4-mediated Runx2 regulation in vivo and in vitro, suggesting miR-exon4 directly targets Nfia and Prkch to regulate Runx2.
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成釉细胞中 Runx2 的消融抑制牙齿发育过程中牙釉质的成熟
影响因子：
4.6
作者：
Chu Q;Gao Y;Gao X;Dong Z;Song W;Xu Z;Xiang L;Wang Y;Zhang L;Li M;Gao Y
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Gao Y
影响因子：
3.6
作者：
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影响因子：
2.7
作者：
Michon, Frederic;Tummers, Mark;Thesleff, Irma
通讯作者：
Thesleff, Irma
DOI：
10.1126/science.1215691
发表时间：
2012-04-13
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Djuranovic S;Nahvi A;Green R
通讯作者：
Green R
影响因子：
4
作者：
Oh, Hyun-Jung;Lee, Hye-Kyung;Park, Joo-Cheol
通讯作者：
Park, Joo-Cheol