【作者】 沈捷；

【导师】 宋咏梅；

【作者基本信息】 北京协和医学院 ， 肿瘤学， 2012， 博士

【摘要】 微小RNA,即nicroRNA(miRNA),是由18到25个核苷酸组成的非编码、小分子单链RNA,具有较高的保守性,几乎所有的多细胞生物都可以表达。MicroRNA以完全或者部分互补结合的方式促进mRNA降解,影响mRNA翻译,对许多基因具有调控作用,在细胞的凋亡、分化及细胞增殖等细胞生物学过程中具有重要作用。近来有越来越多的研究发现,在一些肿瘤组织,特别是在肺癌组织中microRNA有过表达或者低表达的现象,并且找到不同肿瘤组织中的miRNAs表达谱。提示在肿瘤的发生发展过程中,microRNA可能发挥着重要的生物学作用。我们在前期临床研究中发现microRNA-2可以预测小细胞肺癌患者治疗的临床疗效,与预后呈正相关。MicroRNA-2表达水平高的患者具有更好的总体生存率。生存分析表明microRNA-2是小细胞肺癌患者肿瘤复发和生存率的独立预后因子。在本研究中,我们发现通过瞬时转染NCI-H446、NCI-H520、A549以及Anip973肺癌细胞,上调microRNA-2的表达可在体外抑制JNCI-H446、A549以及Anip973肺癌细胞的增殖、侵袭、迁移以及粘附能力。在进一步研究miRNA-2是如何发挥生物学功能的过程中,我们的实验发现miRNA-2能够直接调控PLK1和TGF β1的3’UTR区域,在转录水平影响PLK1和TGF β1的表达,使PLK1和TGF β1的mRNA表达水平下调。考虑到瞬时转染不能完全模拟体内miRNA的生成和加工过程,我们在NCI-H446细胞的基础上构建了miRNA-2持续稳定高表达的细胞系,验证持续稳定高表达miRNA-2对小细胞肺癌细胞系的增殖、侵袭以及迁移能力均有抑制作用,并且能够下调PLK1和’TGFβ1在mRNA和蛋白水平的表达。在miRNA-2引起肺癌细胞系迁移能力下降的机制研究中,我们发现主要是由于miRNA-2的高表达引起TGFβ1的表达下调后,抑制NCI-H446细胞的上皮间质转化,从而在肿瘤转移的初始阶段即抑制了其发生转移。我们运用稳定高表达miRNA-2的细胞系进行动物皮下移植瘤的实验发现,miRNA-2在体内仍能抑制肿瘤细胞增殖、侵袭以及转移能力。而且在高表达miRNA-2的细胞系皮下移植瘤实验中,免疫组化表明PLK1和TGF β1的表达下调。为了验证在临床小细胞肺癌患者中miRNA-2的表达是否也是通过对PLK1和TGFβ1的调节影响患者的生存预后,我们找到前期临床研究中miRNA高表达生存预后好以及miRNAf(?)表达生存预后差病例的病理标本,进行免疫组化检测PLK1,结果提示miRNA-2高表达预后好的病例PLK1低表达。证明人体外实验的结果在人体内可能也以同样的生物学机制发挥作用。综上所述,本研究从临床人体标本中发现有预后和生存指示作用的niRNA-2,并在细胞水平和实验动物水平均阐明了miRNA-2发挥作用的生物学机制,再回到临床的人体标本中证实miRNA-2的靶基因,从而较完整的解释了miRNA-2生物学功能和它对肺癌患者预后影响的原因,为肺癌发生发展的机制研究提供更多研究思路和理论支持。目的研究同一患者分前后两个阶段并在不同体位条件下完成总疗程的治疗中,将两阶段治疗计划相叠加后评价总疗程靶区和危及器官剂量的可行性和临床应用。方法回顾性分析小细胞肺癌的患者10例,每例患者在总疗程中采用不同体位重新定位一次,分两个阶段完成治疗,每个阶段分别制定三维适形计划,疗程总剂量为50Gy。均采用Cadplan R3.1.2三维治疗计划系统完成治疗计划的设计。通过治疗计划系统叠加每例患者总疗程中两个阶段的治疗计划,分析将两个阶段治疗计划叠加的可行性以及第一个阶段与第二阶段治疗计划相叠加后临床应用的可行性。结果1.前后两个阶段气管隆突在左右、前后以及头尾方向运动密切正相关(相关性检验P值均<0.05),证明以气管隆突做为两个阶段的治疗计划叠加标识的可行性；2.前后两个阶段靶区和危及器官体积变化显示除PTV2(计划靶区2)的体积变异大有统计学差异外,其余均无统计学差异,并且各危及器官的剂量学相关性分析中仅脊髓最大剂量在复制计划和原计划之间无相关性外(r=0.337,P=0.341),其余剂量参数均显示出密切正相关。结论运用三维治疗计划系统将同一患者两个阶段不同体位的治疗计划叠加时，在保证与叠加计划等中心点坐标密切相关的解剖结构在两个阶段空间位置一致,以及两个阶段的靶区和危及器官体积无明显差异的前提下；可以对体位不同的两个阶段的治疗计划进行叠加,并在临床上运用叠加计划总体评价全疗程中的靶区和危及器官剂量。更多还原

【Abstract】 MicroRNAs (miRNAs) are evolutionarily conserved, endogenous, small, noncoding RNA molecules of about22nucleotides in length that are discovered in C.elegans about20years ago. MiRNAs spread wide in multicellular organisms and develop in a step-wise process through hairpin precursors cleaved by the dsRNA-specific endonucleases Drosha, Dicer and Argonaute. MiRNAs interact with target messenger RNA at specific sites to induce cleavage or inhibit translation. MiRNAs have been implicated in various important biological processes including embryogenesis, differentiation, organogenesis, cell cycle progression, apoptosis, stress response and metabolism. A number of investigations have demonstrated that altered expression of miRNAs are closely relevant to the clinical malignancies in both solid organs and hematological system, several miRNAs have been implicated in non-small cell lung carcinoma (NSCLC).In previous clinical research, we examined the miRNA expression profiles in human small cell lung cancer(SCLC), and found that downregulation of miRNA-2expression was correlated with poor survival among patients with SCLC.Compared with the patients in low miRNA-2expression group, the patients in high expression group had longer overall survival.NCI-H446, NCI-H520, A549and Anip973were transient transfected with chemically synthesized mature miRNA-2mimic oligonucleotides, introduction of miRNA-2clearly led to a significant decrease in proliferation, invasion and migration in NCI-H446A549and Anip973cell line. Analysis with TargetScan4.0indicates that there is a miRNA-2binding site in the3’UTR regions of the PLK1and TGF-β1.We also measured the mRNA and protein levels of these miRNA-2targeted genes and found that PLK1and TGF-β1expression were substantially reduced following the introduction of miRNA-2into the cells. We further investigated the suppressive role of miRNA-2in SCLC malignancy using animal model. The miRNA-2expression vector was stably transfected into NCI-H446cells and pooled clones were obtained. These pooled clones (NCI-H446-miRNA-2) expressed much higher levels of miRNA-2, exhibited slow-growing capabilities and reduced levels for PLK1and TGF-β1in both mRNA and protein expression. NCI-H446-miRNA-2and NCI-H446-control cells were implanted subcutaneously into the right upper back of nude mice, we measured the volumes of the implanted tumors weekly and found that expression of miR-886-3p significantly suppressed the growth of SCLC tumors in vivo. Interestingly, NCI-H446-miR-886-3p cells and NCI-H446-control cells differed dramatically in their invasive and metastatic potentials. In seeking the the mechanism of migration reduction in lung cancer cell lines, We found that miRNA-2expression downregulated of TGFβ1, which can inhibit epithelial mesenchymal transition in NCI-H446cells. To examine whether dysregulated expressions of miRNA-2targeted genes were linked to clinical outcome of SCLC. Totally,40SCLC specimens with known states of miRNA-2expression were analyzed for PLK1protein staining using an immunohistochemical approach. In the low miRNA-2expression group, strong cytoplasmic staining of PLK1. In contrast, among20samples from high miRNA-2expression group, all most all samples revealed weak or negative staining of PLK1. The difference of Plkl expression between two groups is statistically significant.In this report, we choose miRNA-2which was correlated with survival among patients with SCLC. As to the molecular mechanism(s) by which miRNA-2exerts its role as tumor suppressor, we have shown that miRNA-2negatively regulates PLK.1and TGF-β1, which function in promoting cell proliferation, cell migration, tumor invasive growth and metastasis. Furthermore, we demonstrated that miRNA-2may act as a tumor-suppressor to inhibit SCLC cell proliferation, migration and invasion in both cell and animal models. Therefore, it is reasonably interpreted that during the malignant development of SCLC. The better understanding of SCLC biology would substantially improve the effective therapies available for this challenging disease and promote the development of novel therapeutic agents as well. Objective To analyze the feasibility and the practicability of combining two-course treatment plans of one patient who was localized in different positions during two-course. Methods Ten patients with small cell lung cancer were chosen in the present study. According to the clinical requirement, all patients were localized twice with different positions in total course. Using the Cadplan R3.1.2treatment planning system, conformal planning was designed for each patient in two-course. Total radiation dose was50Gy.The reproductive plans can be applied to evaluate the dosimetric distribution to target volume and the surrounding organs at risk in the total course. Results1.The movements of carina in three-dimensional directions were closely correlated between two courses (P<0.05).2.No significant disparities in target volumes and normal tissue volumes were found except for the volume of PTV2. Except for maximum dose of spinal cord (r=0.337, P=0.341), positive correlations of dosimetric parameters were displayed between reproductive plans and original plans. Conclusions To assure the accuracy of isocenter’s coordinate in combined treatment plans and the positive correlations of dosimetric parameters between reproductive plans and original plans, it is feasible and the practicable to combine two-course treatment plans of one patient who was localized in different position during two-course.更多还原