金葡菌杀白细胞素S组分诱导人髓系白血病细胞凋亡/分化机制及分子靶标研究

Nowadays, chemotherapy is prevalent method for leukemia, but the drugs are limited number, evident toxic side effect and increasing resistance. It is urgent to find new anti-leukemia drugs. Our previous researchs have revealed that the S component of Panton-Valentine leukocidin (LukS-PV) secreted by Staphylococcus aureus can induce apoptosis and differentiation of human myeloid leukemia cells, up-regulate miR-125a expression and activate mitochondrial/MAPK pathway. It was reported that the human complement receptor C5aR is the host target of LukS-PV. So we speculate that LukS-PV may activate mitochondrial/MAPK pathway, induce apoptosis and differentiation of leukemia cells by C5aR and/or miR-125a mediating. In this study, we will over express and inhibit C5aR, miR-125a, mitochondrial and MAPK pathway in both vivo and vitro, to reveal the molecular mechanism of human myeloid leukemia cells apoptosis and differentiation induced by LukS-PV. We will also use the human leukemia primary cells to study the correlation between anti-leukemia effect of LukS-PV and the expression of C5aR、miR-125a, to identify the molecular markers for individualized treatment and efficacy evaluation of LukS-PV. Our research results will lay the foundation for clinical application of LukS-PV. Similar researches have never been published in the domestic and overseas.

目前临床抗白血病药物种类较少、毒副作用较大且耐药性不断增加，因而急需寻找新的靶向药物。我们前期研究发现，金黄色葡萄球菌杀白细胞素S组分（LukS-PV）在体内、外具有抗白血病活性，并可上调miR-125a，活化线粒体和MAPK通路。已有文献报道，补体C5a受体（C5aR）是LukS-PV与细胞膜结合的靶点。据此推测：LukS-PV可能通过C5aR和/或miR-125a 调控线粒体/MAPK通路，促进细胞凋亡和分化，发挥抗白血病效应。本项目拟在体内外，采用过表达和抑制试验分别对C5aR、miR-125a、MAPK和线粒体通路进行干预，以揭示LukS-PV诱导细胞凋亡和分化的机制；采用人白血病原代细胞研究C5aR、miR-125a表达与LukS-PV抗白血病效应的相关性，以探寻LukS-PV个体化治疗和疗效评价的分子标志物。研究结果将为LukS-PV临床应用奠定基础。此类研究国内外未见报道。

细菌毒素具有与靶细胞结合的特异性和细胞毒性，已成为抗肿瘤药物研发新的关注点，一些毒素已被证明具有抗肿瘤活性。LukS-PV是金黄色葡萄球菌分泌的PV-杀白细胞素中的S组分。我们前期研究发现，单组份的LukS-PV可诱导细胞凋亡和分化，有望成为为一种新的白血病靶向治疗药物。本课题旨在揭示LukS-PV诱导白血病细胞凋亡和分化的机制。主要研究内容和结果如下： 1、采用不同浓度LukS-PV作用于AML细胞株，结果显示：LukS-PV处理可使细胞凋亡率增加、细胞周期阻滞、迁移和侵袭能力减弱。细胞表面抗原CD11b和CD14表达增加，吞噬功能增强，向巨噬细胞分化。2、采用转录组学分析和干预试验研究表明：LukS-PV通过激活ERK1/2，JNK ，p38通路，激活下游转录因子c-Fos、c-Jun 和ATF2的磷酸化表达，发挥抗白血病效应。3、构建C5aR过表达和敲低细胞株，结果表明LukS-PV诱导细胞凋亡作用依赖于细胞膜上C5aR表达。采用AML患者的原代细胞进行实验，发现LukS-PV诱导细胞凋亡率与细胞膜上C5aR表达水平呈正相关。4、过表达miR125a-3p可诱导THP-1细胞分化、周期阻滞，并使凋亡率增加，而敲低miR125a-3p，可抑制LukS-PV的抗白血病效应。通过荧光素酶报告实验证实NF1和Bcl2是miR125a-3p的直接靶基因。结果表明LukS-PV通过上调miR125a-3p的表达，下调NF1和Bcl2表达，发挥诱导细胞分化和凋亡作用。5、将HL60（C5aR高表达细胞株）、NB4（C5aR低表达细胞株）分别经尾静脉注入SCID小鼠体内，采用LukS-PV治疗，结果显示，LukS-PV 对HL60细胞的侵袭和浸润抑制作用更加显著，表明LukS-PV在小鼠体内通过靶向C5aR发挥抗白血病效应。.本研究使用体内和体外实验，阐明C5aR、miR-125a-3p和ERK1/2，JNK ，p38信号通路在LukS-PV 诱导白血病细胞凋亡和分化中的作用和机制，为LukS-PV的临床应用奠定了理论基础。在该项目的资助下，我们发表了标注项目资助号的SCI收录论文5篇，中文核心期刊论文1篇。