miR-148b激活线粒体凋亡途径增强非霍奇金淋巴瘤放射敏感性的机制

Non-Hodgkin's lymphoma (NHL) is a heterogeneous group of lymphoid malignancies characterized by an abnormal clonal proliferation of B cells, T cells or NK cells. The incidence of NHL has increased strikingly during the last four decades. Radiotherapy is a well-established approach in the management of NHL across different histological and clinical sub-types. NHLs are known to be radiosensitive tumors. However, local failure rates are in the range of 20% to 30% regardless of the radiation dose delivered, suggesting a subset of resistant disease. Eliminating these radiation-resistant cells may be a key to improve lymphoma radiotherapy..Accumulating evidence indicates that microRNAs (miRNAs) are implicated in cell radiosensitivity. Our previous study has proved that miR-148b is up-regulated after radiation and can enhance the radiosensitivity of NHL by promoting radiation-induced apoptosis. Bioinformatics analysis and preliminary experiments have showed that Bcl-w may be the target gene of miR-148b. Bcl-w protein is an important anti-apoptotic protein in Bcl-2 family, while Bcl-2 family proteins control mitochondrial cytochrome c release and play an extremely important role in the mitochondrial apoptotic pathway. As a result, we propose that miR-148b inhibits the expression of Bcl-w and enhances the radiosensitivity of NHL by activating the mitochondrial apoptotic pathway..In this project, we intend to further validate Bcl-w as the target gene of miR-148b by transient transfection and luciferase reporter assay. Then, we use B-cell and T-cell NHL cell lines as in vitro models, up-regulate or down-regulate miR-148b expression through lentivirus transduction, and detect the corresponding changes of radiosensitivity and mitochondrial apoptotic pathway, including apoptotic index, mitochondrial transmembrane potential change and cytochrome c release, mRNA and protein expression of Bcl-w, Bcl-2, Bcl-XL, Bax, Cyt c, Apaf-1, Caspase-9 and Caspase-3. Furthermore, we up-regulate or down-regulate Bcl-w expression through plasmid transfection to observe whether Bcl-w could reverse the role of miR-148b. Based on in vitro study, we further use nude mice bearing NHL tumors as in vivo models to observe the relationships among miR-148b, Bcl-w, mitochondrial apoptotic pathway and radiosensitivity. The results of in vitro and in vivo experiments will be analyzed together to reveal the molecular mechanism of miR-148b enhancing NHL radiosensitivity..We aim to get the credible evidence that miR-148b takes Bcl-w as target gene and enhances radiosensitivity by activating the mitochondrial apoptotic pathway, and to provide more adequate scientific basis to establish miR-148b as a new radiosensitization target for NHL. At the same time, its mechanism may be extended to other tumors and provide new radiobiological ideas with important scientific significance.

本项目的前期研究已经证明了miR-148b能够促进放射诱导的凋亡，增强非霍奇金淋巴瘤的放射敏感性。生物信息学分析和初步实验表明，Bcl-w可能是miR-148b的靶基因。Bcl-w蛋白是Bcl-2家族中抗凋亡蛋白的重要一员，而Bcl-2家族蛋白在线粒体凋亡途径中发挥极为重要的调控作用。由此，本课题组提出，miR-148b抑制Bcl-w的表达，通过激活线粒体凋亡途径增强非霍奇金淋巴瘤的放射敏感性。本项目拟在非霍奇金淋巴瘤细胞中进一步验证Bcl-w是miR-148b的靶基因，在体外采用慢病毒感染改变miR-148b的表达，检测放射敏感性和线粒体凋亡途径的变化，再用质粒转染改变Bcl-w的表达，观察其能否逆转生物效应，并在体内验证，旨在获得miR-148b以Bcl-w为靶基因，通过激活线粒体凋亡途径增强放射敏感性的可靠证据，为确立miR-148b作为放射增敏治疗的新靶点提供更充分的科学依据。

本项目的前期研究已经证明了miR-148b能够促进放射诱导的凋亡，增强非霍奇金淋巴瘤的放射敏感性。本项目首先分析非霍奇金淋巴瘤Raji细胞照射前后microRNA的差异表达，并利用生物信息学预测其靶基因，从而预测其功能。结果表明，差异表达的miRNA大部分被预测参与细胞周期、凋亡、DNA损伤与修复的生物学过程，提示其可能参与细胞放射敏感性的调控。Bcl-w可能是miR-148b的靶基因。Bcl-w蛋白是Bcl-2家族中抗凋亡蛋白的重要一员，而Bcl-2家族蛋白在线粒体凋亡途径中发挥极为重要的调控作用。这提示，miR-148b可能抑制Bcl-w的表达，通过激活线粒体凋亡途径增强非霍奇金淋巴瘤的放射敏感性。这为我们研究miR-148b的作用机制提供了思路。. 然后我们构建了Bcl-w基因3’UTR质粒和mut Bcl-w基因3’UTR 质粒，转染Raji细胞和HuT-78细胞后，细胞再转染miR-148b模拟剂和抑制剂，双荧光素酶报告基因实验证实Bcl-w是miR-148b的靶基因。. 随后我们利用慢病毒感染构建miR-148b稳定表达或抑制的Raji细胞，并合成了Bcl-w siRNA，构建了Bcl-w表达质粒。细胞功能检测表明，miR-148b过表达能抑制Raji细胞的增殖和克隆形成，而miR-148b低表达能促进Raji细胞的增殖和克隆形成。miR-148b过表达促进Raji细胞的凋亡、线粒体膜电位的下降和细胞色素C的释放，而miR-148b低表达抑制Raji细胞的凋亡、线粒体膜电位的下降和细胞色素C的释放。miR-148b对细胞周期没有明显影响。细胞增殖检测和平板克隆实验表明，miR-148b增强Raji细胞的放射敏感性。Western blot实验表明，miR-148b改变了凋亡相关蛋白的表达。在miR-148b稳定过表达细胞加入Bcl-w表达质粒，或者在miR-148b稳定抑制细胞加入Bcl-w siRNA，均可以逆转miR-148b的生物学功能。这些结果说明，miR-148b以Bcl-w为靶基因，通过激活线粒体凋亡途径增强Raji细胞的放射敏感性。本项目同时发现了钌配合物对肿瘤细胞生长和放射敏感性的调控作用。本项目的结果为确立miR-148b作为放射增敏治疗的新靶点提供更充分的科学依据。