CLDN1上调MMP1的表达促进食管鳞癌细胞TE10侵袭及转移

CLDN1 promotes the proliferation and metastases of esophageal squamous cell carcinoma by up-regulating MMP1

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，mRNA）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。

Abstract:

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 ex－pression of CLDN1 through lentivirus transfection and screening by 1 μg/mL puro，and then the overexpression of CLDN1 was identi－fied by western blot. Furthermore，the proliferation and metastases of indicated group cells were examed by CCK-8 and Transwell as－say respectively. To explore the mechanism underlying，the mRNA microarray was also performed for TE10 with CLDN1 overexpres－sion 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 re－vealed 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 signifi－cant increased in TE10 cells with Lv-CLDN1 transfected and the results was confirmed by qRT-PCR and Western blot. The rescue assay showed that the proliferation and metastases were all inhibited after MMP1 scilencing. Conclusion：CLDN1 promotes the prolif－eration and metastases of TE10 cells by up-regulating MMP1.