MicroRNA-150调控NK/T细胞淋巴瘤放疗敏感性机制研究

Tumor cells to radiation resistence is a common problem in clinical.The miR-15 was demonstrated to be an important regulator of B/T lymphopoiesis.Notably,recent studies suggested that miR-150 may be important in the regulation of hematopoietic injury resulted form chemotherapy and radiotherapy, whether miR-150 affects lymphoma radiation sensitivity? It has not been reported. In our preliminary studies, we found that the radiosensitivity could be regulated by overexpression of miR-150 in Hank-1 and NK-92 cell lines. However, the exact relationship between miR-150 and radiosensitivity is unkown. In this project,we will explore the exact relationship between miR-150 and radiation sensitivity.The mechanism of the regulation of radiosensitivity by miR-150 was analyzed using Hank-1 and NK-92 cell lines. Moreover, we further determine whether PI3K/AKT/mTOR signal, which is an important radiation inducing signal, are regulated by miR-150. We also evaluate the effect of miR-150 expression, AKT level, apoptosis and autophagy and radiation sensitivity in NK/T cell lymphoma by western blot or immunohistochemistry or real-time RT-PCR or immunofluorescence. Moreover, we validate miR-150 enhance radiosensitivity using animal model. Altogether, our project tries to validate our hypothesis that radiosensitivity regulated by miR-150 targeting to AKT control apoptosis and autophagy in NK/T cell lymphoma cells. These findings provide new insights into the molecular functions of miR-150 as a NK/T cell lymphoma sensitizer..

肿瘤细胞辐射抵抗是临床救治中常见的问题。近期发现miR-150调节放化疗药物对造血细胞损伤，miR-150是否会影响淋巴瘤细胞辐射敏感性呢？目前未见相关报道。我们前期实验证实miR-150 可增强NK/T细胞淋巴瘤辐射敏感性，同时AKT、PI3K/Akt/mTOR 信号活性及凋亡与自噬水平在NK/T细胞淋巴瘤细胞辐射效应中均发生改变，经生物信息预测发现AKT2和AKT3等为靶的基因。基于以上，本项目拟在NK/T细胞淋巴瘤细胞上进行miR-150表达的人工干预，验证miR-150 增强肿瘤细胞辐射敏感性是通过靶向AKT，阻截PI3K/Akt/mTOR 信号，诱导淋巴瘤细胞凋亡及自噬来完成的。以证“miR-150-PI3K/Akt/mTOR-凋亡/自噬-辐射敏感性”通路是miR-150调节NK/T细胞淋巴瘤细胞辐射敏感性机制，最终为miR-150在淋巴瘤放疗中的实际应用提供科学依据。

背景：NK/T细胞淋巴瘤辐射抵抗是淋巴瘤治疗的主要挑战。MicroRNA-150在肿瘤中表达异常（包括淋巴瘤）并参与了肿瘤的发生及发展。本研究拟探讨miR-150在调节NK/T细胞淋巴瘤放疗敏感性中作用。.方法：（1）利用RT-PCR技术分析NK/T细胞淋巴瘤组织及细胞系中miR-150表达情况（2）根据组织中miR-150表达量评估其与临床病理的关系（3）利用慢病毒载体技术建立稳定过表达miR-150细胞系（4）体外鉴定细胞增殖能力、克隆形成能力、流式分析细胞凋亡能力、WB分析凋亡蛋白表达（5）荧光素酶报告系统鉴定miR-150的靶点及相关信号通路 （6）体内实验证实免疫缺陷小鼠成瘤及miR-150增敏效应。.结果：miR-150在NK/T细胞淋巴瘤组织及细胞系(NK-92 和Hank-1)中显著低表达，统计学分析证实相比于治疗缓解组，低表达miR-150的患者缓解率低，具有统计学意义（P < 0.05）；过表达miR-150的NK-92 和Hank-1细胞系体外照射后细胞的增殖能力及克隆形成能力较对照组显著降低，具有统计学差异（P < 0.05）；与对照组相比，miR-150过表达组照射后凋亡率明显增加，同时裂解的caspase-3表达量也增加；当AKT2 和AKT3 3'UTR突变时, miR-150不能结合到靶位点，导致荧光素酶活性下降。WB证实与其他组相比，络氨酸激酶抑制剂LY294002 联合miR-150组p-AKT/p-mTOR表达明显下调，同时及其克隆形成能力下降。免疫缺陷小鼠体内实验证实miR-150联合照射组成瘤体积最小。.结论：研究表明miR-150可通过AKT调节通路增强NK/T细胞淋巴瘤放疗敏感性。