miR-450a通过转录后沉默和去甲基化双重调控细胞周期和凋亡促进口腔白斑发展的作用研究

The key of leukoplakia development is the cluster of cells with high proliferation ability and low apoptosis rate. However, the molecular mechanism remains unknown. Our previous studies have found the low expression of miR-450a in high proliferative and low apoptotic leukoplakia cells. The alteration of expression of miR-450a can also regulate the cell proliferation and apoptosis ability. It is speculated that miR-450a can regulate the proliferation and apoptosis of leukoplakia cell. CyclinD1、MDM2 and TET2 are the potential targets of miR-450a. This hints that miR-450a has a dual regulatory function to cell proliferation and apoptosis by post-transcriptional silencing and epigenetic regulation of cyclinD1、MDM2 and TET2. But the fine mechanism needs a further explanation. Base on the high proliferation and low apoptosis/ low proliferation and high apoptosis leukoplakia cell model, this research topic will study the regulation of miR-450a to cyclinD1、MDM2 and TET2 with functional genomics combined with CRISPR/Cas9 gene knockout technology and realize the dual regulation cell cycle and apoptosis; Investigate the feasibility of use miR-450a as an early clinical judgement, prevention and treatment target of oral leukoplakia. The results of our project will reveal the elaborate mechanism that how miR-450a mediate leukoplakia cell acquiring the high proliferation and low apoptosis ability, which has a guidance significance to the clinical prevention and treatment of oral leukoplakia development.

细胞获得高增殖低凋亡特性是白斑发展的关键但机制不明。申请人前期研究发现miR-450a在高增殖低凋亡白斑细胞中低表达且改变miR-450a的表达水平可同时调节细胞的增殖凋亡能力，推测其对白斑细胞增殖凋亡具调控作用。cyclinD1、MDM2和TET2是miR-450a潜在作用靶点，提示miR-450a可能通过对cyclinD1、MDM2和TET2的转录后沉默和表观遗传实现对细胞周期和凋亡的双重调控，但精细机制有待诠释。本课题拟以高增殖低凋亡/低增殖高凋亡白斑细胞为模型，利用功能基因组学结合CRISPR/Cas9基因敲除技术研究miR-450a调控cyclinD1、MDM2和TET2实现对细胞周期和凋亡的双重调控；探索以miR-450a作为口腔白斑发展临床早期判断和防治靶点的可能。结果将揭示miR-450a介导白斑细胞获得高增殖低凋亡特性的精细机制，并对口腔白斑发展的临床防治具有指导意义。

细胞获得高增殖低凋亡特性是白斑发展的关键但机制不明。申请人前期研究发现miR-450a在高增殖低凋亡白斑细胞中低表达且改变miR-450a的表达水平可同时调节细胞的增殖凋亡能力，推测其对白斑细胞增殖凋亡具调控作用。cyclinD1、MDM2和TET2是miR-450a潜在作用靶点，提示miR-450a可能通过对cyclinD1、MDM2和TET2的转录后沉默和表观遗传实现对细胞周期和凋亡的双重调控，但精细机制有待诠释。本课题以高增殖低凋亡/低增殖高凋亡白斑细胞为模型，利用功能基因组学结合CRISPR/Cas9基因敲除技术研究miR-450a调控cyclinD1、MDM2和TET2实现对细胞周期和凋亡的双重调控；探索以miR-450a作为口腔白斑发展临床早期判断和防治靶点的可能。体外研究结果基本证实上述科学假说，但组织样本和动物模型的研究结果呈阴性。调整研究方案，开展了对前期研究提示在口腔白斑发展过程中可能起重要调控作用的其他microRNAs及其调控机制的研究。我们发现miR-223表达与OSCC细胞侵袭和迁移能力负相关。 TCGA数据库数据证实了miR-223表达与OSCC转移之间的关系。功能实验表明，miR-223的过度表达可能降低OSCC细胞的转移能力，而降低其表达水平则导致OSCC转移的增强。生物信息学和双荧光素酶报告基因检测法确定miR-223直接靶向调控TCF7L2。另外，TCF7L2与患者的转移和生存呈负相关。据此我们推测miR-223通过直接靶向TCF7L2并增强Wnt /β-catenin信号通路来调节黏膜上皮恶变后的侵袭和转移。这些发现证明了miR-223在癌变作用中的多种作用。 miR-223可能是潜在的上皮恶变干预靶标。综上，本研究结果揭示了在口腔白斑发展过程中可能起重要调控作用的microRNAs及其调控机制，对口腔白斑发展的临床防治具有指导意义。