摘要
目的预测并验证小鼠mmu-miR-294调控的靶基因,探讨其在肺癌发生发展中的生物学功能。方法生物信息学预测mmu-miR-294可能调控的靶基因金属蛋白酶(MMP3),双荧光素酶检测验证mmu-miR-294调控MMP3的真实性;脂质体2000介导转染mmu-miR-294模拟物进入Lewis(LLC)细胞株,通过Transwell实验检测细胞侵袭、迁移能力的改变。结果重组质粒经XbaⅠ单酶切能获得约5 000 bp和100 bp的酶切片段,阳性克隆测序,双荧光素酶报告基因检测证明合成寡核苷酸链序列插入正确;脂质体2000介导转染mmu-miR-294模拟物,过表达实验组MMP3蛋白水平较对照组明显降低。转染mmu-miR-294模拟物后LLC细胞的侵袭迁移能力显著降低(P<0.01)。结论低表达mmu-miR-294有助于维持LLC的侵袭转移特性,增加其表达水平可以有效抑制LLC的侵袭迁移能力。mmu-miR-294可能通过调控其靶基因MMP3表达而发挥功能。
Objective To predict and validate mmu-miR-294 target gene in mouse lung cancer stem cells, and to investigate its biological functions in the carcinogenesis and development of lung cancer. Methods Bioinformatics analysis predicted that mmu-miR-294 might regulate targeted genes like matrix metalloprotein- ase 3 （MMP3）. The sequence of MMP3 was artificially synthesized, and the seed sequence containing 3＇-UTR was directly inserted to an eukaryotic expression plasmid pGL3-promoter by Xba I digestion. E. coli DH5 alpha was transformed. Positive clones were identified by enzyme digestion and DNA sequencing, and dual luciferase report assay was applied for validation, mmu-miR-294 mimies was transferred into Lewis cells mediated by Lipo- some 2000. The expression level of miR-294 was measured by real-time quantitative PCR, and Western blotting was used to detect the expression level of MMP3 protein. Results The 5 000 bp and 100 bp restrietion frag- ments were obtained after the recombinant plasmids were digested by Xba I . The positive clones were identified by sequencing and dual lueiferase report assay. Compared with the eontrol group, the expression level of MMP3 protein was signifieantly decreased in the mmu-miR-294 mimies transfection group, and the invasion and migra-tion abilities of the Lewis cells was reduced significantly （ P 〈 0.01 ）. Conclusion Up-regulation of mmu- miR-294 can effectively inhibit the invasion and migration of Lewis cells through down-regulating the expression of its target gene MMP3.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2013年第12期1200-1204,共5页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(30772108)
重庆市科委重点攻关项目(CSTC 2011AB5032)~~
