hnRNPK调控细胞自噬相关分子参与髓系白血病耐药机制研究

Imbalance of cell autophagy activity is challenging mechanism for drug resistance in leukemia.There are no reports at home and abroad about hnRNP K regulate autophagic related genes through controlling transcriptic process. Our studies found that hnRNPK and mTOR/ERK/Beclin1 are over-expressed in drug resistant myeloid leukemia patients compared to those drug sensitive ones. Then, the bioinformatics predict hnRNPK could combine with the mRNA of mTOR, ERK and Beclin1. Therefore, we will proceed to validate the correlation about the autophagy, the expression level of hnRNPK/mTOR/ERK/Beclin1 and drug resistant myeloid leukemia from from patients, bone marrow cell lines and animal models.We will futher study whether the hnRNP K could combined with mRNA of mTOR/ERK/Beclin1 through ChIP and three yeast hybrid technology.We will also analyse the relation between production of autophagy and drug resistance by electron microscope and LSCM. All those above would contribute to overcome drug resistance in myeloid leukaemia developing new type of targeted drugs.

细胞自噬活性失衡是导致白血病发生耐药的新机制。hnRNPK是细胞内调节基因转录的"总开关"。本课题组前期研究发现hnRNPK及细胞自噬相关分子 mTOR/ERK/Beclin1与髓系白血病耐药高度相关，生物信息学分析发现hnRNPK可与 mTOR/ERK/Beclin1的RNA结合，由此我们假设：hnRNPK表达上调并激活细胞自噬相关分子的转录，引发细胞自噬活性失衡，最终导致白血病细胞发生耐药。目前国内外尚未见hnRNPK通过调节自噬相关分子致髓系白血病耐药机制的研究。我们拟从髓系白血病患者骨髓原代细胞、细胞株和动物模型中验证hnRNPK/mTOR/ERK/Beclin1的异常表达与细胞自噬及耐药的相关性，染色质免疫共沉淀、酵母三杂交验证hnRNPK与mTOR/ERK/Beclin1的RNA相互结合，电镜及激光共聚焦检测细胞自噬体形成变化，为克服白血病耐药及多靶点药物开发提供新思路。

本年度，课题顺利结题：（1）在临床骨髓原代细胞中验证了hnRNPK的高表达与化疗药物耐药相关，完善了儿童组实验数据。（2）在细胞株水平，证实了hnRNPK的高表达与阿霉素及伊马替尼耐药相关可能与细胞自噬水平增高有关，而调变hnRNPK的表达水平后，进一步证实了hnRNPK表达变化与细胞自噬及药物耐受相关。（3）在自噬调控机制研究中，证实hnRNPK可调控细胞自噬，并可能通过调节细胞自噬的早期阶段Beclin 1复合物参与伊马替尼及阿霉素的耐药。（4）在本年度我们的重点实验为体内部分，体内动物实验进一步验证了体外实验结果：在体内水平，耐药细胞的成瘤速度较野生株增快，耐药株的成瘤体积较野生株增大，荧光镜下观察GFP-LC3II表达变化情况，耐药株成瘤组LC3II的表达强度较野生型细胞株成瘤组及hnRNPK低表达成瘤组增高，这提示耐药株成瘤组自噬水平较野生型细胞株成瘤组及hnRNPK低表达成瘤组增高，这与体外实验水平结果是一致的。由上述结果，我们基本证实了HnRNPK的高表达与阿霉素及伊马替尼耐药相关，并可能是通过调控Beclin1等早期细胞自噬分子参与耐药，相关结果已发表英文SCI文章2篇，中文核心期刊2篇，基本达到了预期课题目标。