miR-22对卵巢癌浸润转移的抑制作用及调控机制研究

【作者】 李俊；

【导师】 鹿欣；

【作者基本信息】 复旦大学 ， 妇产科学， 2011， 硕士

【摘要】 目的探讨miR-22对卵巢癌浸润转移的抑制作用,及其卵巢癌细胞生长、凋亡水平的影响,初步筛选出miR-22发挥作用的潜在靶基因,为调控研究打下基础。方法(1)用Real-time RT-PCR验证miR-22在两对不同侵袭能力的SKOV-3/SKOV-3ip细胞、HO-8910/HO-8910PM细胞中的表达差异,同时选取SKOV-3/SKOV-3ip细胞用于以下实验；(2)在相对低表达miR-22的SKOV-3ip细胞中,用miR-22 Precursor上调其内源性miR-22的表达水平,观察卵巢癌细胞侵袭能力及细胞生长、凋亡水平的改变；在相对高表达miR-22的SKOV-3细胞中,用miR-22 Inhibitor抑制内源性miR-22的表达水平,观察卵巢癌细胞侵袭能力及细胞生长、凋亡水平的改变；(3)生物信息学数据库(TARGETSCAN: http://www.targetscan.org、MIRANDA:http://www.microrna.org和PICTAR: http://www.pictar.mdc-berlin.de)预测miR-22下游与卵巢癌浸润转移转移相关的靶基因；选取其中一个靶基因Tiam1,用双荧光素酶报告基因实验、Western Blot及Real-time RT-PCR共同验证miR-22对Tiam1的调控作用。结果(1)Real-time RT-PCR验证发现,miR-22在高侵袭力的卵巢癌SKOV-3ip细胞和HO-8910PM细胞中均呈相对低表达,其差异方向与我们前期芯片结果一致；(2)在SKOV-3ip细胞中,过表达miR-22则能显著抑制其浸润和迁移能力；在SKOV-3细胞中,降低miR-22表达的能显著促进卵巢癌细胞的浸润和迁移能力；但改变miR-22表达对卵巢癌细胞的生长及凋亡水平无明显影响；(3)生物信息学数据库分析发现,miR-22下游有多个与浸润转移相关的靶基因；对Tiam1进行调控验证后发现,Tiam1是miR-22的直接靶基因；Western Blot及Real-time RT-PCR进一步发现miR-22对Tiaml具有负向调控作用。结论miR-22对卵巢癌浸润转移具有负向调控作用,其功能可能通过靶向Tiam1介导。更多还原

【Abstract】 Objectives. This work is aimed to identify the regulatory effect of miR-22 on cell migration, invasion, growth and apoptosis in ovarian cancer, and to make a general analysis of miR-22’s downstream functional targets with an attempt to gain a better understanding of miR-22’s role in ovarian cancer.Methods. (1)We examined the distinct miRNA expression profiles between paired low-metastatic human serous ovarian cancer cell SKOV-3 and high-metastatic human serous ovarian cell SKOV-3ip using miRNA microarray. Particularly, Real-time RT-PCR was performed to validate miR-22 expression level in paired SKOV-3/SKOV-3ip cells and HO-8910/HO-8910PM cells respectively. (2) Gain-of-function study and loss-of-function study was respectively performed in SKOV-3ip cells, with a relatively higher expression level of miR-22, and SKOV-3 cells, with a relatively lower expression level of miR-22, to monitor the changes in cell invasion and migration, cell growth, and cell apoptosis. (3) Three bioinformatics databases(TARGETSCAN, http://www.targetscan.org, MIRANDA, http://www.microrna.org and PICTAR, http://www.pictar.mdc-berlin.de) was employed to screen the potential targeting genes of miR-22. In particular, we validate the regulatory effect of miR-22 on Tiaml expression by dual-luciferase reporter assay, Western Blot and Real-time RT-PCR.Results. (1) Validation by Real-time RT-PCR revealed that miR-22 was decrease in SKOV-3ip cells and HO-8910PM cells, compared with their counterparts, SKOV-3 cells and HO-8910 cells, respectively. (2) Both gain-of-function and loss-of-function study displayed an negative effect of miR-22 on cell invasion and migration in vitro without significantly affecting cell viability and apoptosis. (3) Subsequent bioinformatics analysis revealed that miR-22 might regulate multiple pro-metastatic genes. Particularly, regulatory study of miR-22 in Tiam1 expression was performed. Our results showed that Tiam1 was a direct target of miR-22, revealed by dual-luciferase reporter assay, and alteration of miR-22 expression had a negative regulatory effect on Tiam1 protein level and mRNA level, demonstrated respectively by Western Blot and Real-time RT-PCR.Conclusions. Taken together, our findings suggested that miR-22 might be involved in inhibiting ovarian cancer metastasis, probably by targeting Tiam1.更多还原