沉默细胞分裂周期相关基因NUF2对食管鳞癌增殖及转移能力的影响及其机制

Effect of silencing cell division cycle associated gene NUF2 on proliferation and metastasis of esophageal squamous cell carcinoma and its mechanism

目的探讨沉默细胞分裂周期相关基因(NUF2)对食管鳞癌细胞增殖及转移能力的影响以及其相关的分子机制。方法回顾性分析癌症基因组图谱(TCGA)数据库中食管癌患者原发病灶及其癌旁组织的NUF2基因表达差异,采用反转录聚合酶链反应(RT-PCR)方法分析食管鳞癌细胞系的NUF2基因表达,设计靶向NUF2基因的沉默RNA(siRNA)序列,构建慢病毒载体,Celigo法、噻唑蓝(MTT)法检测细胞增殖,荧光激活细胞分选法(FACS)法检测细胞凋亡,Transwell方法检测细胞迁移和侵袭能力,蛋白质印迹法(Western blot)检测蛋白表达,慢病毒转染过表达变化最明显的五个下游基因,再检测细胞增殖、迁移和侵袭能力的变化。组间比较采用t检验。结果MTT法表明,沉默NUF2基因后细胞增殖低于对照组(0.33±0.01比0.55±0.07,t=25.90,P<0.01),Transwell法提示沉默NUF2基因后细胞迁移低于对照组(KYSE-150:9.00±1.67比36.00±4.84,t=35.53,P<0.01;TE-1:52±1.86比246.00±9.79,t=45.73,P<0.01),沉默NUF2的食管癌细胞中、过表达SNAI1基因后,增殖、迁移和侵袭能力又有所恢复。结论NUF2基因在食管鳞癌中高表达,沉默其表达能够通过下调SNAI1基因来抑制细胞增殖、迁移和侵袭。

Objective To investigate the effect of silencing cell division cycle associated geneNUF2 gene on the proliferation and metastasis of esophageal squamous cell carcinoma and its mechanism.Methods We retrospectively analyzed the differences of NUF2 gene expression in the primary lesions of esophageal cancer patients and their adjacent normal tissues in the Cancer Genome Atlas(TCGA)database.The NUF2 gene expression was measured in esophageal squamous cell carcinoma cell lines by reverse transcription-polymerase chain reaction(RT-PCR).The small interfering RNA(siRNA)sequence targeting the NUF2 gene was designed and synthesized.The transfection efficiency was observed by fluorescence microscope after 72 h,and the expression level of NUF2 gene and protein was detected by RT-PCR and Western blotting at 72 h after transfection.Celigo cell counting method and the methyl thiazolyltetrazolium(MTT)method were used to detect tumor cell proliferation.Fluorescence activated cell sorting(FACS)method was used to detect cell apoptosis and Transwell method to detect cell migration and invasion ability.Western blotting was used to detect the expression change of proteins in classical downstream pathways.Lentivirus infection was used to overexpress the downstream genes with the most obvious change and then changes in cell proliferation,migration and invasion ability were examined.The t test was used to compare data between two groups.Results MTT method showed that the cell proliferation ability decreased significantly after silencing the NUF2 gene(0.33±0.01 vs.0.55±0.07,t=25.90,P<0.01).Transwell method indicated that cell migration was significantly decreased after silencing NUF2 gene(t=35.53,45.73,P<0.01).After the SNAI1 gene was overexpressed by lentivirus infection,the ability of proliferation,migration and invasion was restored.Conclusion NUF2 gene is up-regulated in esophageal squamous cell carcinoma.Silencing its expression can inhibit cell proliferation,migration and invasion by down-regulating the expression of SNAI1 gene.