miR-320c靶向FADD基因抑制肺腺癌细胞凋亡促进癌细胞的迁移和侵袭

miR-320c promotes the migration and invasion of lung adenocarcinoma cells through inhibiting apoptosis via targeting FADD gene

目的:研究miR-320c调控肺腺癌细胞迁移和侵袭的分子机制。方法:Real-time PCR方法检测miR-320c在肺腺癌细胞株A549和SPC-A-1中的表达情况。通过质粒转染构建miR-320c过表达和敲低的肺腺癌细胞株,采用Transwell实验检测miR-320c对肺腺癌细胞迁移和侵袭的影响。通过流式细胞技术检测肺腺癌细胞的凋亡率,免疫共沉淀检测凋亡通路关键复合体的表达判断细胞发生凋亡的情况。生信分析预测miR-320c的下游靶基因,并通过双荧光素酶实验验证。结果:与正常肺上皮细胞相比,miR-320c在肺腺癌细胞中表达升高。过表达miR-320c能够促进肺腺癌细胞的迁移和侵袭,而敲低miR-320c则抑制细胞的迁移和侵袭。进一步实验证实miR-320c通过抑制肺腺癌细胞凋亡发挥其促进迁移和侵袭的作用。荧光素酶报告基因系统验证了miR-320c直接靶向凋亡通路的关键基因FADD;FADD的上调可以有效逆转miR-320c mimic诱导的促进迁移和侵袭作用。结论:miR-320c通过靶向FADD基因抑制肺腺癌细胞凋亡促进癌细胞的迁移和侵袭。

Objective:To investigate the molecular mechanism of miR-320c regulating the migration and invasion of lung adenocarcinoma cells.Methods:Real-time PCR was used to detect the expression of miR-320c in lung adenocarcinoma cell line A549 and SPC-A-1.The overexpressed and knockdown miR-320c lung adenocarcinoma cell lines were constructed by plasmid transfection,and detecting miR-320c effect on the migration and invasion of lung adenocarcinoma cells by Transwell assay.The apoptotic rate of lung adenocarcinoma cells was detected by flow cytometry,and the expression of key complexes in apoptotic pathway was detected by Co-IP to determine the occurrence of cell apoptosis.The downstream target genes of miR-320c were predicted by bioanalysis and verified by dual luciferase assay.Results:Compared with normal lung epithelial cells,the expression of miR-320c was increased in lung adenocarcinoma cells.Overexpression of miR-320c promoted migration and invasion of lung adenocarcinoma cells,while knockdown miR-320c could inhibit its migration and invasion.Further experiments confirmed that miR-320c could promote the migration and invasion of lung adenocarcinoma cells by inhibiting apoptosis.The luciferase reporter gene system verified that miR-320c directly targeted FADD,a key gene in apoptosis pathway,the up-regulation of which could effectively reverse the promotion of migration and invasion induced by miR-320c mimic.Conclusion:miR-320c promoted the migration and invasion of lung adenocarcinoma cells by targeting FADD gene to inhibit apoptosis.