Lnc RNA KCNQ1OT1靶向miR-506-3p调控舌鳞状细胞癌细胞增殖、侵袭和迁移的机制研究

Research on the mechanism of Lnc RNA KCNQ1OT1 on the proliferation, invasion and migration of tongue squamous cell carcinoma cells through targeted regulation of miR-506-3p

目的探讨长链非编码RNA(Lnc RNA)KCNQ1OT1通过靶向调控miR-506-3p对舌鳞状细胞癌(tongue squamous cell carcinomas,TSCC)细胞增殖、侵袭和迁移作用机制。方法实时荧光定量PCR(RT-qPCR)检测正常牙龈上皮细胞(human gingival epithelial cells,HGE)及TSCC细胞(CAL27、SCC9和SCC15细胞系)中KCNQ1OT1和miR-506-3p表达水平。以CAL27细胞为研究对象,构建KCNQ1OT1低表达或miR-506-3p高表达的CAL27细胞,四甲基噻唑蓝染色法(MTT)检测细胞增殖,Transwell检测细胞迁移和侵袭,蛋白印迹(Western blot)检测细胞周期蛋白D1(CyclinD1)、基质金属蛋白酶2(MMP2)、MMP9和β-连环蛋白(β-catenin)表达水平。双荧光素酶报告基因实验验证KCNQ1OT1与miR-506-3p调控关系。结果与HGE细胞比较,TSCC细胞系CAL27、SCC9和SCC15中KCNQ1OT1表达升高(P<0.05),miR-506-3p表达降低(P<0.05)。KCNQ1OT1低表达或miR-506-3p高表达后,CAL27细胞存活率、迁移和侵袭细胞数及CyclinD1、MMP2和MMP9蛋白表达降低(P<0.05)。KCNQ1OT1低表达还降低了CAL27细胞中β-catenin蛋白表达(P<0.05)。miR-506-3p低表达降低了KCNQ1OT1低表达对CAL27细胞存活率、迁移和侵袭细胞数及CyclinD1、MMP2、MMP9和β-catenin蛋白表达的影响(P<0.05)。结论KCNQ1OT1低表达可抑制TSCC细胞增殖、迁移和侵袭,其作用机制与上调miR-506-3p表达和抑制Wnt/β-catenin信号通路的激活有关。

Objective To investigate the mechanism of Lnc RNA KCNQ1 OT1 on the proliferation,invasion and migration of tongue squamous cell carcinoma(TSCC)cells through targeted regulation of miR-506-3 p.Methods The expression levels of KCNQ1 OT1 and miR-506-3 p in normal gingival epithelial cells(HGE)and TSCC cell lines(CAL27,SCC9 and SCC15)were detected by RT-qPCR.CAL27 cells were used as research objects.CAL27 cells with KCNQ1 OT1 low expression or miR-506-3 p high expression were constructed.MTT was used to detect cell proliferation.Transwell was used to detect cell migration and invasion.Western blot was used to detect the expression of Cyclin D1,MMP2,MMP9 andβ-catenin protein.The double luciferase reporter gene experiment was applied to verify the regulatory relationship between KCNQ1 OT1 and miR-506-3 p.Results Compared with HGE cells,KCNQ1 OT1 expression of TSCC cell lines CAL27,SCC9 and SCC15 increased(P<0.05),and miR-506-3 p expression decreased(P<0.05).After KCNQ1 OT1 low expression or miR-506-3 p high expression,CAL27 cell survival rate,number of migrating and invading cells,and expression of CyclinD1,MMP2 and MMP9 protein decreased(P<0.05).KCNQ1 OT1 low expression also reducedβ-catenin protein expression in CAL27 cells(P<0.05).The low expression of miR-506-3 p mitigated the effects of KCNQ1 OT1 low expression on the survival rate of CAL27 cells,the number of migrating and invading cells,and the expression of CyclinD1,MMP2,MMP9 andβ-catenin protein(P<0.05).Conclusion The low expression of KCNQ1 OT1 can inhibit the proliferation,migration and invasion of TSCC cells.The mechanism of action is related to the up-regulation of miR-506-3 p expression and the inhibition of Wnt/β-catenin signaling pathway activation.