microRNA-374b及其靶基因MCP-1调节恶性肿瘤细胞生长转移机制研究

Monocyte chemoattractant protein (MCP-1) participates in prolifiration, metasitasis and immunosuppression of many malignancies, but the underlying mechanism still remains unclear. At the same time, the contribution of microRNA in tumor continuously confirmed. In previous studies, we found that all of the NCI-60 cells could express MCP-1, and the level of MCP-1 had a positive correlation with the malignancy degree of tumor cells, a negative correlation with the prognosis；the dual luciferase experiment confirmed that 3’-UTR of MCP-1 and miR-374b could be combined. Accordingly, we propose the hypothesis: miR374b in malignancy tumor tissue could regulate tumor cells proliferation and metastasis by its target MCP-1. In this study, two breast carcinoma cell lines MCF-7 and MDA-MB-231 will be selected, which have different malignancy degree and different expression levels of MCP-1. Through interference/over-express MCP-1 and miR374b genes, dual luciferase experiment and so on, to observe the relationship of miR374b and MCP-1, their influence on cell proliferation and metastasis. After obtaining conclusive evidence about miR374b and MCP-1, we will using the methods of real-time PCR and gene microarray to analysis the signaling pathways and mechanism involved in. At last, we will test this mode of action in other tumors. Through this research we will explore the possibility and feasibility of the application about using microRNA to inhibiting the infiltration and metastasis of maligant.

单核细胞趋化因子（MCP-1）参与多种恶性肿瘤细胞增殖转移与抗肿瘤免疫，但其机制尚未明确。而microRNA 在肿瘤发生发展中作用不断得到证实。课题组前期研究发现MCP-1在NCI-60系统60种肿瘤细胞株中均有一定程度表达，且表达水平与细胞恶性度成正相关，与患者预后成负相关；实验证实MCP-1 3’-UTR端可与miR-374b结合。据此提出假说：肿瘤局部miR-374b可通过其靶基因MCP-1调控肿瘤细胞增殖转移。本研究将首先采用MCF-7和MDA-MB-231两种恶性度不同且MCP-1表达水平亦不同的乳癌细胞系，通过过表达、干扰miR-374b和MCP-1的转录/表达水平等细胞及动物学实验，研究二者关系及对肿瘤细胞增殖转移的影响，之后通过基因芯片等技术进一步研究其可能涉及的信号通路及机制，最后在其它肿瘤中进行验证。以期探索应用microRNA分子抗肿瘤转移的可能性和可行性。

MCP-1在恶性肿瘤发生发展中的作用得到不断的关注。在本次的研究中我们证实了MCP-1本身即具有促进乳腺恶性肿瘤细胞增殖转移的作用；miR-374b作为MCP-1的上游基因，可通过下调乳腺恶性肿瘤细胞中MCP-1的表达抑制肿瘤细胞的增殖转移并促进细胞凋亡。之后我们将构建的MCP-1过表达和干扰表达的小鼠在小鼠乳腺垫处原位种植，证实了MCP-1在小鼠在体实验中仍然具有促进肿瘤发展的能力,而miR-374b则具有相反的作用。最后对所有构建的稳转细胞行全基因表达谱分析，并对差异表达基因real time PCR验证，结果显示CENPQ (Homo sapiens centromere protein Q, mRNA:NM_018132), FOSL2 (FOS-like antigen 2), HBA1 (Homo sapiens hemoglobin subunit alpha 1 , mRNA:NM_000558), SLPI （Homo sapiens secretory leukocyte peptidase inhibitor, mRNA ：NM_003064），ST3GAL5（Homo sapiens ST3 beta-galactoside alpha-2,3-sialyltransferase 5, transcript variant 1, mRNA ：NM_003896）和OAS2（Homo sapiens 2'-5'-oligoadenylate synthetase 2, 69/71kDa, transcript variant 1, mRNA ：NM_016817）可能是microRNA-374B-MCP-1通路的下游基因。该问题的阐明，解答了MCP-1在乳腺恶性肿瘤增殖转移中的作用，为MCP-1单抗应用于恶性肿瘤免疫靶向治疗提供理论依据。