【作者】 杨健；

【导师】 向荣； 李娜；

【作者基本信息】 南开大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 在肿瘤的发生发展过程中,肿瘤相关巨噬细胞(Tumor associated macrophages,TAMs)的浸润和表型的变化,尤其是由M1型向M2型转化是肿瘤恶变和发展的重要标志之一。对于这一变化过程,相关信号通路和分子机制仍处于研究之中,但其机制还有待进一步探讨。据报道,microRNA在单核巨噬细胞的分化过程中参与了其成熟和功能的变化,这是一种不编码蛋白的小RNA,文献表明该小RNA在各物种都存在并起着重要的功能,同时在肿瘤的发生和发展过程中也起着重要作用,能够作为抑癌基因或原癌基因影响着肿瘤细胞增殖、侵袭和转移等各项功能。基于此,我们将microRNA的功能和肿瘤相关巨噬细胞在肿瘤的发展和转移中的作用联系起来,着力于研究microRNA在肿瘤相关巨噬细胞表型变化及其对肿瘤迁移和侵袭能力的影响。首先,我们以小鼠的原代巨噬细胞、小鼠白血病来源的巨噬细胞系RAW264.7以及人单核巨噬细胞系U937作为巨噬细胞模型,同时以人乳腺癌细胞系MDA-MB-231和小鼠乳腺癌细胞系4T1作为乳腺癌细胞模型,研究了乳腺癌微环境对巨噬细胞中的microRNA表达的影响。由于Fra-1基因对于肿瘤相关巨噬细胞的表型极化有重要影响,因此我们通过TargetScan软件预测了可能结合Fra-1mRNA3’UTR区的microRNA,发现miR-19a-3p这一在脊椎动物中广泛保守的microRNA在Fra-1的3’UTR区有潜在的结合位点。进一步的研究表明乳腺癌细胞培养液上清的刺激可以下调miR-19a-3p在巨噬细胞中的表达,伴随Fra-1这个在乳腺癌发展和转移起中起促进作用的原癌基因蛋白表达的升高。我们通过将合成的miR-19a-3p mimic转染巨噬细胞,发现其能够抑制Fra-1的表达。通过双荧光报告实验的分析,我们揭示miR-19a-3p是通过结合Fra-1mRNA3’UTR区而抑制该基因表达的。同时伴随Fra-1下游基因Stat3、p-Stat3和VEGF表达的下降。更重要的是,当巨噬细胞过表达miR-19a-3p时,其在体外促进肿瘤细胞侵袭和迁移的能力明显下降。通过Real-time PCR、流式细胞实验及细胞因子蛋白芯片分析,我们发现miR-19a-3p不仅能够抑制巨噬细胞的M2表面标志分子的表达,而且抑制M2型巨噬细胞相关细胞因子,如IL-4, IL-6,IL10等的分泌。基于这些体外实验的结果,我们使用了人工合成的、可用于动物实验的miR-19a-3p agomir,通过瘤内注射的方法,在肿瘤组织内过表达miR-19a-3p。经多次注射后,取出肿瘤分析组织内肿瘤相关巨噬细胞的表型变化,通过免疫组化染色、免疫荧光染色和流式细胞技术分析,我们发现注射miR-19a-3p agomir组小鼠相比注射对照agomir组小鼠的肿瘤组织内的M2型肿瘤相关巨噬细胞的比例明显下降。更为重要的是,通过HE染色,我们发现注射miR-19a-3p agomir组小鼠的肺部转移结节数量和大小均明显小于注射对照agomir组小鼠,这表明miR-19a-3p能够在瘤内充分抑制肿瘤细胞的肺转移。不过,通过活体成像技术实时监测小鼠的原位肿瘤的生长,我们发现瘤内注射miR-19a-3p并不影响其生长能力。总而言之,我们一系列的体外细胞实验和动物体内实验表明miR-19a-3p是一个肿瘤相关巨噬细胞M2表型极化的抑制分子,而诱导miR-19a-3p的低表达对于巨噬细胞中Fra-1的表达上调和M2型极化起重要作用。因此,miR-19a-3p具有潜在的临床治疗价值,有希望结合纳米颗粒靶向技术应用到临床治疗中,为治疗乳腺癌提供了可供选择的分子。更多还原

【Abstract】 One of the hallmarks of malignancy is the polarization of tumor-associated macrophages (TAMs) from a pro-immune (M1-like) phenotype to an immune-suppressive (M2-like) phenotype. However, the molecular basis of the process is still unclear. MicroRNA (miRNA) is comprised by a group of small, non-coding RNAs which are broadly expressed by a variety of organisms and involved in such cell behaviors as suppression or promotion of tumorigenesis. Based on these prvious findings, we tried to explore the relationship between the function of miRNA and TAMs in tumor progression, and how miRNAs regulate the phenotype of TAMs and the progress of tumor metastasis. Firstly, we used mouse primary macrophages, mouse leukemia macrophage cell line RAW264.7, human mononuclear macrophage cell ine U937as macrophage model, and human breast cancer cell line MDA-MB-231, mouse breast cancer cell line4T1were used as breast cancer cell model. We tested how the tumor microenvironment regulates the miRNA expression of TAMs. Based on the function of Fra-1in regulating the polarization of TAMs, we predicted the potential targeting miRNAs of Fra-1with Target Scan programme. Here, we demonstrate that miR-19a-3p, broadly conserved among vertebrates, has potential binding site in3’UTR of Fra-1and is down-regulated in RAW264.7and U937macrophage cells of M2phenotype by conditioned medium of4T1and MDA-MB-231breast tumor cells:This down-regulation correlated with an increased expression of the Fra-1gene which was reported to play the role of a pro-oncogene by supporting invasion and progression of breast tumors. We found significant up-regulation of miR-19a-3p in RAW264.7macrophages, following the transfection with a synthesized miR-19a-3p mimic, which leads to significant suppression in expression of Fra-1gene. Additionally, we confirmed that miR-19a-3p bind to site the3’UTR of Fra-1with a psiCHECK luciferase reporter system. Furthermore, transfection of RAW macrophage cells with the miR-19a-3p mimic decrease expression of VEGF, STAT3and p-STAT3, which are downstream gene of Fra-1signaling. By real-time PCR, we found that miR-19a-3p down-regulates the M2markers of macrophages as well as suppressing the M2cytokines, such as IL-4, IL-6and IL10.Based on the in vitro results, we synthesized miR-19a-3p agomir and applied it to animal experiment by injecting it intratumor to overexpress miR-19a-3p in TAMs in vivo. After6times of injection, we removed the primary tumor to analysis the phenotype of TAMs in tumor tissue by IHC, IF and flow cytometry. We found that miR-19a-3p down-regulated the proportion of M2macrophages within tumor. Most importantly, by HE staining, we found that the number and size of metastasis nudes were down-regulated by miR-19a-3p, indicating that the metastatic capacity of4T1breast tumor cells on lung tissues was significantly impaired. However, the growth rate of primary tumor was not affected. Taken together, these findings indicated that miR-19a-3p is capable of inhibiting the M2phenotype of TAMs and the low expression of this microRNA plays an important role in up-regulating the expression of Fra-1and induction the polarization of M2macrophage. Therefore, combined with nanoparticle-mediated delivery approach, it is possible that miR-19a-3p could also become a potentially useful therapeutic target for the treatment of breast cancer.更多还原