miR-216b-5p对喉癌TU686细胞增殖、迁移和侵袭的影响及其机制

Effect of miR-216b-5p on proliferation,migration and invasion of laryngeal cancer TU686 cells and its mechanism

目的:探讨miR-216b-5p对喉鳞状细胞癌(LSCC)TU686细胞增殖、迁移、侵袭和凋亡的影响,分析其在喉癌细胞中的靶基因及其可能的作用机制。方法:TU686细胞分为对照组和miR-216b-5p组,对照组细胞通过慢病毒感染无义序列,miR-216b-5p组细胞通过慢病毒感染过表达miR-216b-5p。采用实时荧光定量PCR(RT-qPCR)法检测2组细胞中miR-216b-5p表达水平,克隆形成实验检测2组细胞克隆形成率,细胞划痕实验检测2组细胞划痕愈合率,Transwell实验检测2组细胞中侵袭细胞数,流式细胞术检测2组细胞凋亡率。通过TargetScan网站预测miR-216b-5p的潜在靶基因,采用RT-qPCR法检测靶基因mRNA表达水平,双荧光素酶报告基因实验检测miR-216b-5p与靶基因间的靶向关系。结果:慢病毒感染后,与对照组比较,miR-216b-5p组细胞中miR-216b-5p表达水平明显升高(P<0.01)。与对照组比较,miR-216b-5p组细胞克隆形成率和划痕愈合率明显降低(P<0.01),侵袭细胞数明显减少(P<0.01),细胞凋亡率明显升高(P<0.01)。TargetScan网站预测,自噬相关基因5(ATG5)为miR-216b-5p潜在的靶基因,与对照组比较,miR-216b-5p组细胞中ATG5 mRNA表达水平明显降低(P<0.01)。双荧光素酶报告基因实验证实ATG5与miR-216b-5p之间存在靶向关系。结论:miR-216b-5p通过抑制喉癌细胞的增殖、迁移、侵袭和诱导细胞凋亡进而发挥抑癌作用,其机制可能与靶向调节ATG5的表达水平有关。

Objective:To investigate the effect of miR-216b-5p on the proliferation,migration,invasion and apoptosis of laryngeal squamous cell cancer(LSCC)TU686 cells,and to analyze the target genes in laryngeal cancer cells and its possible mechanism.Methods:The TU686 cells were divided into control group and miR-216b-5p group;the cells in control group were infected with the nonsense sequence through lentivirus,and the cells in miR-216b-5p group were infected with overexpressed miR-216b-5p by lentivirus.The expression levels of miR-216b-5p in the cells in two groups were detected by real-time fluorescence quantitative PCR(RT-qPCR)method,the clone formation rates of the cells in two groups were detected by clone formation assay,the scratch healing rates of cells in two groups were detected by cell scratch assay,the number of invasion cells in two groups was detected by Transwell assay,and the apoptotic rates of cells in two groups were detected by flow cytometry.The TargetScan website was used to predict the potential target genes of miR-216b-5p.The expression levels of target gene mRNA were detected by RT-qPCR method.The targeting relationship between miR-216b-5p and the target gene was detected by dualluciferase assay.Results:After lentivirus infection,compared with control group,the expression level of miR-216b-5p in the cells in miR-216b-5p group was significantly increased(P<0.01).Compared with control group,the clone formation rate and the scratch healing rate of the cells in miR-216b-5p group were significantly decreased(P<0.01),the number of invasion cells was significantly decreased(P<0.01),and the apoptotic rate was significantly increased(P<0.01).Targetscan website predicted that autophagy related gene 5(ATG5)was the potential target gene of miR-216b-5p;compared with control group,the expression level of ATG5 mRNA in the cells in miR-216b-5p group was significantly decreased(P<0.01).The dual-luciferase assay proved that there was a targeting correlation between ATG5 and miR-216b-5p.Conclusion:MiR-216b-5p can inhibit the proliferation,migration,and invasion and induce apoptosis of laryngeal cancer cells to exert a tumor suppressor effect,and its mechanism may be related to the targeted regulation of the expression level of ATG5.