miR-9/CXCR4抑制急性单核细胞白血病细胞侵袭性生长的机制研究

急性单核细胞白血病SHI-1细胞具有较强的体外移行、粘附、裸鼠体内成瘤及致白血病能力。申请者发现下调CXCR4表达能显著抑制SHI-1侵袭性生长，miR芯片等生物学信息分析发现低表达CXCR4的SHI-1高表达miR-9。有研究认为miR-9靶向降解CXCR4，起抑制肿瘤作用。据此推测miR-9可通过CXCR4抑制SHI-1细胞侵袭性生长。本项目将检测miR-9与CXCR4 3`UTR的靶向结合，通过miR-9启动子甲基化及去甲基化探讨miR-9低表达机制；在SHI-1中过表达miR-9，检测CXCR4下游信号通路改变，通过增殖、粘附、移行、裸鼠体内成瘤及浸润实验观察miR-9对SHI-1细胞侵袭能力的影响；过表达miR-9后，用mRNA芯片等探讨miR-9其它可能靶基因。本项目将揭示miR-9/CXCR4通路抑制SHI-1细胞侵袭性生长的机制，为急性白血病病理机制及治疗研究提供新思路。

SHI-1 cells, an acute monocytic leukemia cell line, had highly aggressive infiltration characters, demonstrated with strong migration, adhesion, tumorigenicity ability in vitro and in nude mice. Our previous studies had shown that when the expressions of CXCR4 in SHI-1 cells were knocked-down, the aggressive infiltration characters of SHI-1 cells were significantly inhibited. microRNA chip and real-time PCR shown that the miR-9 in SHI-1 cells with low expression of CXCR4 were significantly higher than SHI-1 cells. Other studies had shown that miR-9, which could target to the CXCR4, had the functions of tumor inhibition. But the roles of miR-9/CXCR4 in acute leukemia were not well elucidated. We hypothesis that miR-9 could inhibited the aggressive growth of SHI-1 cells both in vitro and in vivo via targeting CXCR4. In this project, luciferase assay would be used to investigate the targeted combination between the seed sequence of miR-9 and 3`-UTR district of CXCR4 mRNA; miR-9 DNA promoter methylation and demethylation would be used to explain the concrete mechanism of the lower expression of miR-9 in SHI-1 cell. The expression of miR-9 in SHI-1 cells would be up-regulated by lentivirus vector. Western blotting would be used to detect the protein of CXCR4 downstream signal pathway such as ERK1, ERK2, AKT, P-AKT, P38MAPK and NF-kB. The proliferation, adhesion, invasion and apoptosis capacity of SHI-1/miR-9 cells would be investigated in vitro to verify the roles of miR-9 in SHI-1 cells. SHI-1/miR-9 cells would be inoculated to the nude mice in subcutaneous or by tail vein, to investigate the growth model of SHI-1 cells with higher miR-9 in vivo. The changes of mRNA in the SHI-1/miR-9 cells would be analyzed by mRNA chips, others possible targets genes of miR-9 would be ascertained by bioinformatics, real-time PCR, luciferase assay, and so forth. The project will reveal concrete mechanisms of miR-9/CXCR4 pathways in the malignant behavior of SHI-1 cells, and would provide new ideas and targets for the study and treatment of leukemia infiltration.