miR-875-5p通过靶向USF2抑制胃癌细胞的增殖、迁移和侵袭

目的:探讨miR-875-5p对胃癌细胞增殖、迁移和侵袭的影响及其机制。方法:采用qPCR法检测胃癌细胞BGC-823、HGC-27、MGC-803、SGC-7901、AGS、MKN-45和胃黏膜上皮细胞GES-1中miR-875-5p的表达水平。利用脂质体转染技术,分别将miR-875-5p模拟物/抑制剂(mimic/inhibitor)及其阴性对照质粒(miR-NC/Anti-miR-NC)转染至AGS细胞/MKN-45细胞,构建过表达/抑制miR-875-5p的细胞模型,空白对照组(Control组)不转染。通过CCK-8、克隆形成、Transwell等实验分别检测miR-875-5p表达变化对细胞增殖、克隆形成、迁移和侵袭的影响。采用双荧光素酶报告基因实验验证miR-875-5p与上游刺激因子2(USF2)的靶向关系,WB实验验证miR-875-5p对USF2的调控作用并检测USF2蛋白的表达。构建MKN-45细胞裸鼠移植瘤模型,验证miR-875-5p过表达对MKN-45细胞成瘤能力的影响。结果:miR-875-5p在6种胃癌细胞中表达水平显著低于胃黏膜上皮细胞GES-1(均P<0.01)。与Control组和miR-NC组相比,miR-875-5p mimic组AGS细胞的增殖、克隆形成率、迁移和侵袭细胞数,以及USF2蛋白的表达均显著降低(P<0.05或P<0.01);miR-875-5p inhibitor组MKN-45细胞的增殖、克隆形成率、迁移和侵袭细胞数,以及USF2蛋白的表达均显著提高(P<0.05或P<0.01)。双荧光素酶报告基因实验证明,miR-875-5p能够直接靶向USF2基因。体内成瘤实验结果表明,过表达miR-875-5p显著抑制MKN-45细胞移植瘤的生长(均P<0.01)。结论:miR-875-5p通过靶向USF2抑制胃癌细胞的增殖、迁移和侵袭。

miR-875-5p inhibits the proliferation, migration and invasion of gastric cancer cells by targeting USF2

Objective: To investigate the effects of miR-875-5p on proliferation, migration and invasion of gastric cancer (GC) cells and its mechanism. Methods: The expression level of miR-875-5p was detected by qPCR in GC cell lines (BGC-823, HGC-27,MGC-803, SGC-7901, AGS, MKN-45) and gastric epithelial GES-1 cells. MiR-875-5p mimics/inhibitors or their negative control plasmids (miR-NC/anti-miR-NC) were transfected into AGS or MKN-45 cells by liposome transfection technique to construct miR-875-5p overexpression or knockdown cell model. Cells in blank control group (Control group) were not transfected. The effects of miR-875-5p on cell proliferation, clone formation, migration and invasion were detected by CCK-8 assay, colony formation assay and Transwell assay, respectively. The targeting relationship between miR-875-5p and upstream stimulatory factor 2 (USF2) was verified by dual-luciferase reporter gene assay. The regulation of miR-875-5p on USF2 as well as the expression of USF2 protein was confirmed by WB assay. After the construction of MKN-45 cell transplanted tumor model in nude mice, the effect of miR-875-5p overexpression on tumorigenesis of MKN-45 cells was detected. Results: The expression level of miR-875-5p in 6 GC cells was significantly lower than that in gastric epithelial GES-1 cells (all P<0.01). Compared with Control group and miR-NC group, the proliferation, clone formation rate, number of migrated and invaded cells, and USF2 protein expression level in AGS cells were significantly decreased in miR-875-5p mimic group (P<0.05 or P<0.01), while those were significantly increased in MKN-45 cells of miR-875-5p inhibitor group (P<0.05 or P<0.01). Dual-luciferase reporter gene assay demonstrated that miR-875-5p could directly target USF2 gene. In vivo tumorigenesis experiment results showed that overexpression of miR-875-5p significantly inhibited the growth of MKN-45 cell transplanted tumors (all P<0.01). Conclusion: miR-875-5p inhibits proliferation, migration and invasion of GC cells by targeting USF2.