摘要
目的:研究紧密连接蛋白-1(tight junction protein 1,CLDN1)在食管鳞癌细胞TE10中的生物学功能及机制。方法:体外构建CLDN1过表达病毒,按感染复数(multiplicity of infection,MOI)=20的比例感染食管鳞癌细胞TE10。通过蛋白质印迹(Western blot)检测CLDN1过表达效果。分别采用细胞增殖及毒性检测试剂(cell counting Kit-8,CCK-8)检测细胞增殖能力,肿瘤细胞迁移及侵袭实验(Transwell)检测细胞迁移及侵袭能力变化。通过基因芯片分析过表达CLDN1对TE10基因表达谱的影响,分析下游靶基因并通过Western blot和回复实验研究CLDN1对MMP1的表达调控。结果:与NC组相比,过表达组TE10细胞中基质金属蛋白酶1(matrix metallopeptidase-1,MMP1)蛋白表达为(2.68±0.10),明显升高(P<0.05);CCK-8实验结果表明,过表达组细胞在24、48、72 h时的吸光度明显高于阴性对照组(P<0.05);迁移实验结果显示过表达组TE-10细胞迁移细胞数明显高于阴性对照组[(54.8±3.4)vs.(24.6±2.1)];侵袭实验结果显示,过表达组TE-10细胞侵袭数目高于阴性对照组[(34.5±2.6)vs.(11.5±1.6)(P<0.05)。信使核糖核酸(messenger RNA,m RNA)芯片分析发现,过表达CLDN1后TE10细胞的MMP1基因表达明显升高(P<0.05),定量聚合酶链反应(polymerase chain reaction,PCR)及Western blot证实CLDN1能够促进MMP1的表达;功能回复实验结果显示,干扰MMP1能够抑制CLDN1对TE10细胞的增殖和侵袭转移能力的促进作用。结论:CLDN1可通过上调MMP1的表达促进食管鳞癌TE10细胞的增殖和侵袭转移,发挥癌基因的功能。
Objective:To investigate the role of CLDN1 on the proliferation and metastases of esophageal squamous cell carcinoma(ESCC)cell line TE10 and the underlying mechanism. Methods:ESCC cell line TE10 was cultured and constructed with stable expression of CLDN1 through lentivirus transfection and screening by 1 μg/m L puro,and then the overexpression of CLDN1 was identified by western blot. Furthermore,the proliferation and metastases of indicated group cells were examed by CCK-8 and Transwell assay respectively. To explore the mechanism underlying,the m RNA microarray was also performed for TE10 with CLDN1 overexpression to screen target gene downstream. At last,the rescue assay was performed to confirm the mechanism. Results:Compared with that of negative group,matrix metallopeptidase1(MMP1)expression was significantly increased(2.68±0.10)(P〈0.05). CCK-8 array revealed that the proliferation of TE10 with Lv-CLDN1 transfected was significantly attenuated [(34.5±2.6)vs.(11.5±1.6)](P〈0.05).Transwell assay confirmed that the cell numbers of TE10 with Lv-CLDN1 transfected penetrated the membrane were obviously more than those of negative control [(54.8±3.4)vs.(24.6±2.1)](P〈0.05). The microarray revealed that MMP1 expression was significant increased in TE10 cells with Lv-CLDN1 transfected and the results was confirmed by q RT-PCR and Western blot. The rescue assay showed that the proliferation and metastases were all inhibited after MMP1 scilencing. Conclusion:CLDN1 promotes the proliferation and metastases of TE10 cells by up-regulating MMP1.
作者
曾义波
吴剑
李俊
张大伟
任德莲
戴天阳
Zeng Yibo;Wu Jian;Li Jun;Zhang Dawei;Ren Delian;Dai Tianyang(Department of Cardiothoracic Surgery,the Affiliated Hospital of Southwest Medical Universit)
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2018年第5期712-716,共5页
Journal of Chongqing Medical University
基金
2017年自贡市重点科技计划资助项目(编号:2017SF03)
关键词
食管鳞癌
紧密连接蛋白1
基质金属蛋白酶-1
肿瘤增殖和转移
esophageal squamous cell carcinoma
tight junctionprotein 1
matrix metallopeptidase-1
cell proliferation and migration