microRNA调控上皮-间充质转变在石棉致恶性间皮瘤中的作用机制研究

Malignant mesothelioma（MM） is an aggressive tumor that develops in the parietal pleura or peritoneum and is generally attributable to asbestos exposure. Once detected its rapidly fatal, with the median surviral time between 4-18 months. The molecular mechanism of asbestos-induced malignant mesothelioma is not clear. microRNA（miRNA） are new molecular targets which play fundamental roles in various biological processes through regulation of gene expression at the level of post-transcription.In our previous study, we found 11 miRNA related with epithelial-mesenchymal transition(EMT）may be involved in malignant mesothelioma. In this study, aberrantly expressed microRNAs which screened from previeous microRNA expression profiles will be determined in MM cells and asbestos treated mesothelial cells; MM cells and asbestos treated mesothelial cells will be transfected with miRNA mimics or inhibitors,miRNA and further cell proliferation, apoptosis, clonogenicity, cell migration and invasion assaies, clonogenicity and nude mouse transplantation tumor experiment, epithelial-mesenchymal transition Markers and its transcriptioanl factors will be determined; The putative miRNA targets related with EMT were predicted by using miRNAs databases and verified. The results of this study will clear-out the MiRNA mechanism of MM occur and provide theoretical basis of MM diagnosis and therapeutic.

恶性间皮瘤（Malignant Mesothelioma, MM）是一种起源于胸膜腔或腹膜腔上皮或间皮组织的侵袭性肿瘤，其发病主要与石棉接触有关。石棉所致恶性间皮瘤的分子机制并不清楚。我们前期工作筛选到的在恶性间皮瘤细胞中异常表达的microRNA(miRNA)中，有11个可能与上皮-间充质转变（epithelial-mesenchymal transition，EMT）相关。本项目拟研究石棉处理间皮细胞、恶性间皮瘤细胞与正常间皮细胞间的miRNA的异常表达，逆转这些异常表达的miRNA，研究其对细胞恶性表型、EMT表型、EMT调控转录因子的影响以及对miRNAs进行精确靶点验证，以明确异常表达miRNA调控EMT在石棉所致间皮瘤中的作用机制。该项目将进一步阐明石棉诱导恶性间皮瘤的分子机制，并为恶性间皮瘤的生物标志物早期诊断与分子靶向治疗提供重要的理论依据和前期工作。

恶性间皮瘤（Malignant Mesothelioma, MM）是一种起源于胸膜腔或腹膜腔上皮或间皮组织的侵袭性肿瘤，其发病主要与石棉接触有关。石棉所致恶性间皮瘤的分子机制并不清楚。.本项目研究了间皮瘤细胞NCI-H2052、NCI-H2452与正常间皮细胞Met-5A中hsa-miR-155-5p和hsa-miR-324-5p、hsa-miR-34b-5p 、hsa-miR-34c-5p、hsa-miR-28-5p、hsa-miR-30d-5p共6种miRNA的表达情况及石棉致miRNA改变的时间-反应关系，恶性间皮瘤细胞与石棉处理细胞中E-cadherin、VIMENTIN、TWIST1基因与蛋白变化；并研究了NCI-H2452细胞转染miRNA-30d类似物miRNA mimics后细胞增殖、周期与凋亡、迁移、侵袭等表型的变化、EMT表型及EMT调控转录因子Twist的表达变化；在NCI-H2452细胞上转染miRNA-28 mimics，观察恶性间皮瘤细胞在细胞迁移、侵袭等表型的变化；用软件预测miRNA作用靶点，对其进行精确靶点验证，观察miR-28靶基因IMPDH1变化，利用双荧光素酶报告系统验证靶基因，并研究了抑制IMPDH1基因对恶性间皮瘤细胞迁移、侵袭的的影响。研究发现，在间皮瘤细胞、石棉处理的间皮细胞中均出现miR-30d表达下调，miR-30d可以抑制NCI-H2452细胞的增殖、迁移、侵袭，促进细胞凋亡，并逆转EMT标志蛋白的表达变化；miR-28可以抑制NCI-H2452细胞迁移、侵袭，其机制可能是通过抑制IMPDH1基因。因此，本项目结果提示，miR-30d、miR-28可能被定义为肿瘤抑制miRNA，其下调可能促进胸膜间皮瘤的发生发展和石棉致癌，miR-28通过调节IMPDH1基因可能可以抑制MM的发生发展。miR-30d、miR-28具有作为恶性间皮瘤诊断和肿瘤治疗靶点的潜在用途。