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 metalloproteinase 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 XbaⅠ digestion. E. coli DH5 alpha was transformed. Positive clones were identified by e

nzyme digestion and DNA sequencing

and dual luciferase report assay was applied for validation. mmu-miR-294 mimics was transferred into Lewis cells mediated by Liposome 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 restriction fragments were obtained after the recombinant plasmids were digested by XbaⅠ. The positive clones were identified by sequencing and dual luciferase report assay. Compared with the control group

the expression level of MMP3 protein was significantly decreased in the mmu-miR-294 mimics transfection group

and the invasion and migration 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.