1.辽宁省肿瘤医院;2.辽宁省肿瘤医院 病理科;3.辽宁省肿瘤医院 头颈外科
liaoning cancer hospital and institution
目的 探究lncRNA NEAT1对喉鳞状细胞癌细胞迁移和侵袭的影响,并进一步探究miR-429/ZEB1轴在其中发挥的作用。方法慢病毒转染建立稳定敲减lncRNA NEAT1的喉鳞状细胞癌细胞,通过RT-qPCR检测细胞lncRNA NEAT1表达以验证转染效率,通过Transwell实验检测细胞迁移和侵袭能力,通过Western blot检测细胞EMT相关蛋白N-cadherin、Vimentin、Slug和Snail表达。通过生物信息学分析miR-429与lncRNA NEAT1和ZEB1 mRNA间的潜在结合位点,并通过双荧光素酶报告基因实验验证miR-429与lncRNA NEAT1以及miR-429与ZEB1 mRNA结合。将miR-429抑制剂转染敲减lncRNA NEAT1的LSCC细胞,通过Western blot检测细胞ZEB1表达。进一步检查敲减ZEB1对抑制miR-429的敲减lncRNA NEAT1的LSCC细胞迁移、侵袭和EMT的影响。结果 敲减lncRNA NEAT1降低LSCC细胞lncRNA NEAT1表达,抑制细胞迁移和侵袭并下调细胞N-cadherin、Vimentin、Slug和Snail表达。miR-429与lncRNA NEAT1和ZEB1 mRNA间存在潜在结合位点,且miR-429与lncRNA NEAT1以及miR-429与ZEB1 mRNA结合。抑制miR-429逆转了敲减lncRNA NEAT1对LSCC细胞ZEB1表达的抑制作用。此外,敲减ZEB1逆转了抑制miR-429对敲减lncRNA NEAT1的LSCC细胞迁移和侵袭的促进作用及EMT相关蛋白N-cadherin、Vimentin、Slug和Snail表达的上调作用。结论lncRNA NEAT1促进LSCC细胞迁移和侵袭,其机制可能与海绵化miR-429上调ZEB1表达促进LSCC细胞EMT有关。
Objective To explore the effect of lncRNA NEAT1 on the migration and invasion of laryngeal squamous cell carcinoma cells, and further exploring the role of miR-429/ZEB1 axis in this process. Methods Slow virus transfection established stable knockdown of lncRNA NEAT1 in laryngeal squamous cell carcinoma cells. The expression of lncRNA NEAT1 was detected by RT-qPCR to verify transfection efficiency. Cell migration and invasion ability were detected by Transwell assay, and the expression of EMT related proteins N-cadherin, Vimentin, Slug, and Snail was detected by Western blot. Analyze the potential binding sites between miR-429 and lncRNA NEAT1 and ZEB1 mRNA through bioinformatics, and validate the binding of miR-429 to lncRNA NEAT1 and miR-429 to ZEB1 mRNA through dual luciferase reporter gene experiments. Transfect LSCC cells with lncRNA NEAT1 knockdown using miR-429 inhibitor and detect ZEB1 expression by Western blot. Further examination of the impact of knocking down ZEB1 on the migration, invasion, and EMT of LSCC cells that inhibit miR-429 knockdown lncRNA NEAT1. ResultsKnocking down lncRNA NEAT1 reduces the expression of lncRNA NEAT1 in LSCC cells, inhibits cell migration and invasion, and downregulates the expression of N-cadherin, Vimentin, Slug, and Snail in cells. There are potential binding sites between miR-429 and lncRNA NEAT1 and ZEB1 mRNA, and miR-429 binds to lncRNA NEAT1 and miR-429 with ZEB1 mRNA. Inhibiting miR-429 reversed the inhibitory effect of knocking down lncRNA NEAT1 on ZEB1 expression in LSCC cells. In addition, knockdown of ZEB1 reversed the inhibitory effect of miR-429 on the migration and invasion of LSCC cells with knockdown lncRNA NEAT1, as well as the upregulation of EMT related proteins N-cadherin, Vimentin, Slug, and Snail expression. Conclusion LncRNA NEAT1 promotes migration and invasion of LSCC cells, and its mechanism may be related to the upregulation of ZEB1 expression by spongy miR-429, which promotes EMT in LSCC cells.