应用miR-17-92 cluster 诱导的多潜能造血干细胞免疫治疗肿瘤的研究

The miR-17-92 cluster plays an important regulatory role in the hematopoietic stem cells proliferation, differentiation and apoptosis. We have recently reported that the miR-17-92 cluster promotes expansion of pluripotent hematopoietic stem cells (PHSC) in mice. Cell lines derived from these miR-17-92 cluster-overexpressing mice are capable of myeloid and lymphoid lineage differentiation in vitro, and recapitulate the normal lymphoid phenotype when transplanted to immunodeficient NOD/SCID mice. This is the theory basis for us to study a new approach for tumor immunotherapy using these PHSC induced by over-expressing miR-17-92 cluster. We will inject pMSCV2.1, a retroviral vector encoding the miR-17-92 cluster or an empty vector control into Balb/C mice bearing orthotopically implanted breast cancer FE1.2 cell line. The efficiency of the induced PHSC to suppress tumor growth will be evaluated by tumor volume. The flow cytometric analysis of isolated splenocytes and bone marrow cells from miR-17-92 over-expressing or vector control breast cancer Balb/C mouse model will be performed to determine whether miR-17-92 cluster increase a fraction of cells with PHSC surface markers. The miR-17-92 cluster induced PHSC and control splenocytes will be classified into different subgroups according to their surface makers; the respective subgroups will be injected into Balb/C mice. Three months post injection, the expanded subgroups in mice will be confirmed as miR-17-92 cluster target cell(s). Microarray analysis will be performed to discover the miR-17-92 cluster target genes using the RNA from miR-17-92 cluster target cells and control splenocytes, which have identical PHSC surface markers. The potential genes will first be confirmed by using a luciferase gene reporter system, then the cDNA(s) will be transfected into miR-17-92 cluster-induced PHSC cell line (YJ3) to determine potential gene(s) function. The gene(s) that halt cells proliferation, promote their differentiation and apoptosis are directly targeted by miR-17-92 cluster. The cytokine secreted by the miR-17-92 cluster target cells will be analyzed by cytokine arrays and ELISAs. This project will demonstrate the anti-tumor function and molecular mechanism of PHSC induced by the miR-17-92 cluster, and investigate their possible use as an anti-tumor immunotherapy strategy.

miR-17-92 cluster 在造血干细胞的生长,分化和凋亡过程中具有重要调节作用。我们已报道了miR-17-92 cluster能诱导小鼠多潜能造血干细胞增生, 增生的干细胞能在体内外生长分化成各种成熟血细胞。本课题以此为理论依据, 以Balb/C小鼠乳腺癌模型为研究平台，观察表达miR-17-92 cluster的反转录病毒载体对移植瘤生长的影响。流式细胞仪分析肿瘤模型小鼠增生的脾细胞;根据细胞表面标记,将增生的干细胞分成的不同细胞亚群注射Balb/C小鼠,体内筛选、确定miR-17-92 cluster作用的靶细胞,并检测靶细胞体外抗肿瘤活性。基因芯片分析具有相同细胞表面标记的靶细胞和正常脾细胞RNA,揭示靶基因;再经cDNA转染增生的干细胞确认靶基因功能。揭示miR-17-92 cluster诱导的多潜能造血干细胞增生作用的靶细胞和靶基因,阐述其抗肿瘤作用的机制：

miR-17-92 cluster 在造血干细胞的生长,分化和凋亡过程中具有重要调节作用。我们已报道了miR-17-92 cluster能诱导小鼠多潜能造血干细胞增生, 增生的干细胞能在体内外生长分化成各种成熟血细胞。本课题以此为理论依据, 以Balb/C小鼠乳腺癌模型为研究平台，观察表达miR-17的反转录病毒载体对移植瘤生长的影响。我们发现：miR-17-92 cluster中的miR-17亚单位对小鼠移植肿瘤的生长和转移具有抑制作用并探讨了其机制。调节miR-17-92 cluster表达的上游基因-过氧化物酶体增殖物活化受体beta/delta（Proliferator-activated Receptor beta/delta，PPAR beta/delta）的分子功能也将被研究。关于对与miR-17-92 cluster的促进子PPRE特异性结合的PPAR beta/delta 的进一步研究表明：PPAR beta/delta具有促进乳腺癌细胞株及慢性淋巴细胞白血病（CLL）患者的B淋巴细胞在培养环境不利于细胞生长的不适条件下继续生存的能力。PPAR beta/delta调节B淋巴细胞中的信号转导过程，可以是通过改变细胞胆固醇代谢水平和调节细胞因子，如I型干扰素的细胞内信号转导的结果。同时我们发现PPAR beta/delta拮抗剂具有作为抗白血病信号转导调节剂的治疗活性。揭示了PPAR beta/delta的分子生物功能及肿瘤免疫调控机制，为治疗癌症的研究提供肿瘤细胞免疫和分子生物学理论基础。