RNA去甲基化酶FTO介导的MALAT1调控TRIM28参与胶质瘤替莫唑胺耐药的机制研究

Temozolomide (TMZ) resistance affects the chemotherapy effect of glioma. Previously, we found that TMZ-resistant U251 cells had high expression of TRIM28, and knockdown of TRIM28 reduced TMZ resistance, which suggested that TRIM28 is associated with TMZ resistance. Further studies showed that RNA demethylase FTO was highly expressed in TMZ-resistant glioma cells asl well, and could affect TRIM28 expression. MeRIP method found that FTO could regulate the m6A level of MALAT1, and bioinformatics showed that both MALAT1 and TRIM28 had miR-491 response elements. Therefore, it is speculated that high expression of FTO leaded to RNA-demethylation and expression of MALAT1, then MALAT1 competitive binded to miR-491 and decreased inhibition of TRIM28, and involved in glioma TMZ resistance. In this project, we first observed the expression and significance of FTO/MALAT1/TRIM28 and TMZ-resistant in glioma cells at the cellular level, and explored the regulatory mechanism between the them; and then the value of the three molecules with TMZ resistance was studied in nude mice glioma in situ model; finally, the correlation of these molecules with TMZ resistance was verified at the clinical patient level. Our work may help to identify reveal the molecular mechanism of glioma TMZ resistance and provide new ideas and evidence for its prevention and treatment.

替莫唑胺（TMZ）耐药影响胶质瘤化疗效果。申请者预实验发现TMZ耐药U251细胞TRIM28高表达，敲低TRIM28减轻TMZ耐药，提示TRIM28与TMZ耐药相关。进一步研究显示RNA去甲基化酶FTO在耐药胶质瘤细胞也高表达，且能影响TRIM28表达。MeRIP方法发现FTO能调控MALAT1-m6A，生物信息学显示MALAT1与TRIM28均有miR-491应答元件。由此推测FTO高表达导致MALAT1去甲基化和表达增加，竞争结合miR-491并降低其对TRIM28的抑制，参与胶质瘤TMZ耐药。本项目先在细胞水平观察FTO/MALAT1/TRIM28耐药胶质瘤细胞的表达和意义，探讨三者之间的调控机制；再在裸鼠脑胶质瘤原位模型研究上述三个分子在TMZ耐药中的价值；最后在临床患者水平验证这些分子与TMZ耐药的相关性。本研究旨在揭示胶质瘤TMZ耐药的分子机制，为其防治提供新思路和和证据。

替莫唑胺是临床上应用最为广泛的胶质瘤化疗药物，它能够通过血脑屏障进入颅内发挥药效。然而在替莫唑胺的临床应用中，胶质瘤固有的或获得性的耐药使得部分患者使用替莫唑胺化疗效果欠佳。阐明胶质瘤替莫唑胺耐药发生发展的机制，寻找新的有效防治措施和新靶标，对进一步提高胶质瘤的治疗水平具有重要的基础和临床意义。本研究在U251细胞（U251）和TMZ耐药的U251细胞（U251TR）中观察两种细胞中FTO、TRIM28和MALAT1表达情况，以及表达水平的相关性，明确耐药细胞株中三者的表达情况；其次，根据FTO在两种细胞中的表达情况，分别在U251细胞中上调FTO表达，在U251TR细胞中下调FTO表达，观察FTO对U251细胞耐药的影响，明确FTO对细胞耐药的影响；再在干预FTO表达的U251细胞中，观察TRIM28和MALAT1表达情况，以及MALAT1的m6A水平变化，明确FTO对TRIM28表达调控的机制。研究结果显示：①与U251相比，U251TR中FTO、TRIM28和MALAT1高表达；②FTO能够影响U251细胞的替莫唑胺耐药；③FTO调控TRIM28表达，其机制可能与FTO调控MALAT1的m6A修饰有关。以上研究揭示FTO在胶质瘤TMZ耐药中的调控作用及其机制，为肿瘤耐药的防治提供新思路与实验依据。