敲减MET表达对喉癌Hep-2细胞增殖、迁移及5-FU和顺铂敏感性的影响

Effects of MET knockdown on proliferation,migration and sensitivity to 5-FU and cisplatin of laryngeal cancer Hep-2 cells

目的:探究敲减间质表皮转化因子(MET)表达对人喉鳞状细胞癌(LSCC)Hep-2细胞生长、迁移及5-氟尿嘧啶(5-FU)和顺铂敏感性的影响。方法:通过基因表达综合数据库(GEO)及癌症基因组图谱(TCGA)数据库数据分析人LSCC组织中MET mRNA的表达水平;常规培养正常人支气管上皮细胞16HBE与人LSCC细胞Hep-2、KBV200和TU212,采用qPCR法和WB法检测16HBE、Hep-2、KBV200和TU212细胞中MET基因和蛋白的表达水平。用LipofectamineTM3000将MET敲减质粒(si-Met)和对照质粒(si-NC)转染至Hep-2细胞中,分为空白对照组,si-NC组和si-Met组。采用MTT法、流式细胞术、划痕愈合实验分别检测各组Hep-2细胞的增殖、迁移能力、周期分布以及对5-FU和顺铂的敏感性。结果:数据库数据分析显示LSCC组织中MET mRNA呈高表达(P<0.05),Hep-2、KBV200和TU212细胞中MET mRNA和蛋白的表达水平也均明显高于16HBE细胞(均P<0.01)。敲减MET表达后,Hep-2细胞中METmRNA及蛋白表达水平均明显降低(P<0.01或P<0.001)、细胞增殖活力显著下降(P<0.0001)、G0/G1期细胞数量明显升高(P<0.0001)、S期细胞数量明显降低(P<0.0001)。敲减MET表达后,不同浓度5-FU或顺铂对细胞增殖的抑制率均显著升高、药物半数抑制浓度(IC50)均降低(均P<0.0001),划痕愈合率明显降低、迁移能力下降(均P<0.05)。结论:MET在人LSCC组织和细胞中呈高表达,敲减MET可有效抑制Hep-2细胞中MET的表达,抑制细胞增殖、迁移能力,使其周期阻滞于G1期,增强Hep-2细胞对5-FU和顺铂的敏感性。

Objective:To investigate the effects of mesenchymal to epithelial transition factors(MET)knockdown on the proliferation,migration,and sensitivity to 5-FU and cisplatin in human laryngeal squamous cell carcinoma(LSCC)Hep-2 cells.Methods:The expression of MET mRNA in LSCC tissues was analyzed using data from the Gene Expression Omnibus(GEO)and The Cancer and Tumor Gene Atlas(TCGA).Human normal bronchial epithelial cells(16HBE)and human LSCC cells(Hep-2,KBV200 and TU212)were routinely cultured,and the expression levels of MET in these cells were detected by qPCR and WB assay.The MET knockout plasmid(si-Met)and control plasmid(si-NC)were transfected into Hep-2 cells using LipofectamineTM 3000,and the cells were divided into blank control group,si-NC group and si-Met group.The proliferation,migration,cell cycle distribution,and sensitivity to 5-FU and cisplatin of Hep-2 cells in each group were detected by MTT assay,flow cytometry and scratch assay,respectively.Results:Database analysis showed high expression of MET mRNA in LSCC tissue(P<0.05).The mRNA and protein expression levels of MET in Hep-2 cells,KBV200 cells and TU212 cells were significantly higher than those in 16HBE cells(all P<0.01).After MET knockdown,the mRNA and protein levels of MET in Hep-2 cells were significantly reduced(P<0.01 or P<0.001),cell proliferation activity was significantly decreased(P<0.0001),the number of G0/G1 phase cells was significantly increased(P<0.0001),and the number of S phase cells was significantly reduced.Additionally,after MET knockdown,the inhibitory rates of cell proliferation by different concentrations of 5-FU or cisplatin were significantly enhanced,and the half maximal inhibitory concentration(IC50)was reduced(all P<0.0001).The scratch healing rate and migration ability were significantly reduced(all P<0.05).Conclusion:MET is highly expressed in human LSCC tissues and cells.Knocking down MET can effectively inhibit the expression of MET in Hep-2 cells,suppress cell proliferation and migration ability,arrest the cell cycle in G1 phase,and enhance the sensitivity of Hep-2 cells to 5-FU and cisplatin.