论文部分内容阅读
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 the target gene regulated by mmu-miR-294 in mice and explore its biological function in the development and progression of lung cancer. Methods The bioinformatics method was used to predict the expression of MMP-3, a target gene regulated by mmu-miR-294, and the duality luciferase assay was used to confirm the authenticity of MMP3 by mmu-miR-294. 294 mimics into Lewis (LLC) cell lines, and the changes of cell invasion and migration were detected by Transwell assay. Results The recombinant plasmid was digested with Xba I to obtain about 5 000 bp and 100 bp fragments. The positive clones were sequenced and the double-luciferase reporter gene was used to confirm the insertion of the synthetic oligonucleotide. After transfection with mmu-miR-294 mimics, the level of MMP3 protein in the overexpression group was significantly lower than that in the control group. The invasion and migration ability of LLC cells after transfection of mmu-miR-294 mock was significantly reduced (P <0.01). Conclusion The low expression of mmu-miR-294 can help maintain the invasion and metastasis of LLC. Increasing the expression level of MMP-2 can effectively inhibit the invasion and migration of LLC. mmu-miR-294 may function by regulating its target gene MMP3 expression.