高级检索

    m6A甲基转移酶METTL3对SCAPs定向分化的调节机制研究

    Studies on the regulatory mechanism of m6A methyltransferase METTL3 on the directed differentiation of SCAPs

      摘要
    • 摘要:
      目的： 探讨甲基转移酶样蛋白3（METTL3）介导的N6-甲基腺苷（m6A）修饰对根尖牙乳头干细胞（SCAPs）向成牙本质细胞分化的调节作用机制以及METTL3介导的m6A对SCAPs中核转录因子I-C（NFIC）表达的调控作用。
      方法： 分离SCAPs，采用流式细胞术检测细胞表面标记分子，茜素红染色和油红O染色检测细胞的分化能力。采用瞬时转染法构建沉默METTL3的SCAPs作为SCAPs METTL3（–）组，并设置空载对照组SCAPs（Ctrl）组。2组细胞矿化诱导1周和2周后，Western blotting分析成牙相关蛋白碱性磷酸酶（ALP）、骨钙蛋白（OCN）、牙本质涎磷蛋白（DSPP）的表达。比色法分析2组中m6A RNA甲基化水平。MeRIP-qPCR和Western blotting分析NFIC的mRNA和蛋白表达水平。
      结果： 流式细胞术分析分离的SCAPs表面标记分子CD44（89.8%）、CD90（92.0%）表达呈阳性，茜素红染色和油红O染色镜下见矿化结节和脂滴形成。SCAPs METTL3（–）组METTL3蛋白水平明显低于SCAPs（ctrl）组（P ＜ 0.01）。矿化诱导1周和2周后，SCAPs METTL3（–）组矿化量及ALP、OCN、DSPP蛋白水平低于SCAPs（ctrl）组（P ＜ 0.05 ~ P ＜ 0.01）。比色法结果显示， SCAPs METTL3（–）组中m6A mRNA甲基化水平明显低于SCAPs（Ctrl）组（P ＜ 0.01）。Western blotting结果显示，SCAPs METTL3（–）组中NFIC的蛋白表达水平明显低于SCAPs（Ctrl）组（P ＜ 0.01）。
      结论： METTL3介导的m6A修饰可以调节SCAPs向成牙本质细胞分化以及能够通过NFIC发挥调节SCAPs的作用。

       

      Abstract:
      Objective To investigate the mechanism of the regulatory effect of methyltransferase-like protein 3 (METTL3) mediated N6-methyladenosine (m6A) modification on the differentiation of stem cells from apical papilla (SCAPs) to dentin cells and the regulatory effect of METTL3-mediated m6A on the expression of nuclear factor I-C (NFIC) expression in SCAPs.
      Methods SCAPs were isolated, and flow cytometry was applied to analyze cell surface marker molecules, alizarin red staining and oil red O staining were used to assess the differentiation capacity of the cells. A transient transfection method was employed to construct SCAPs silenced for METTL3, which was set as the SCAPs METTL3(–) group, and an empty vector control group SCAPs (Ctrl) group was established. After 1 week and 2 weeks of mineralization induction, Western blotting was performed to analyze the expression of tooth formation-related proteins, including alkaline phosphatase (ALP), osteocalcin (OCN), and dentin sialophosphoprotein (DSPP). The m6A RNA methylation levels in the two groups were measured by colorimetry. MeRIP-qPCR and Western blotting were utilized to evaluate the mRNA and protein expression levels of NFIC.
      Results Flow cytometry analysis showed that surface marker molecules CD44 (89.8%) and CD90 (92.0%) were positive in the isolated SCAPs. Mineralization nodules and lipid droplet formation were observed under the microscope by alizarin red staining and oil red O staining. The METTL3 protein level in the SCAPs METTL3(–) group was significantly lower than that in the SCAPs (ctrl) group (P ＜ 0.01). After 1 week and 2 weeks of mineralization induction, the mineralization amount and ALP, OCN, DSPP protein levels in the SCAPs METTL3(–) group were lower than those in the SCAPs (ctrl) group (P ＜ 0.05 to P ＜ 0.01). The results of colorimetry showed that the m6A mRNA methylation level in the SCAPs METTL3(–) group was significantly lower than that in the SCAPs (Ctrl) group (P ＜ 0.01). The results of Western blotting indicated that the protein expression level of NFIC in the SCAPs METTL3(–) group was significantly lower than that in the SCAPs (Ctrl) group (P ＜ 0.01).
      Conclusions METTL3-mediated m6A modification can regulate the differentiation of SCAPs into odontoblasts as well as being able to play a role in regulating SCAPs through NFIC.

       

      • HTML全文
      • 参考文献(23)
      • 相关文章
      • 施引文献
    • 资源附件(0)

    /

    返回文章
    返回