Lang Yuanyuan, Meng Jie, Song Xiaomeng, Chen Xiaojun
Department of Clinical Laboratory, Tianjin Children's Hospital, Tianjin 300074, China.
Department of Immunology, Basic Medical College, Tianjin Medical University, Tianjin 300070, China.
Zhongguo Fei Ai Za Zhi. 2015 Feb;18(2):92-7. doi: 10.3779/j.issn.1009-3419.2015.02.08.
EFEMP1, a member of fibulin family proteins, is a very important extracellular matrix protein which is involved in cell metabolism and its role in tumor occurrence and progression is still poorly understood. The aim of this study is to investigate the functional effect and mechanism of EFEMP1 in lung cancer cell growth and invasion.
EFEMP1 expression in lung cancer cells was determined by Western blot. The promoter methylation status of EFEMP1 was detected by methylation-specific PCR (MSP). After transfection of control or EFEMP1 vector in lung cancer cells, the ability of colony formation and invasion was detected by colony formation experiment and matrigel invasion method. Western blot and real-time PCR were used to detect matrix metalloproteinase-7 (MMP-7) expression. Luciferase assay was used to detect expression of MMP-7 reporter construct transfected with or without EFEMP1 in lung cancer cells.
Western blot result showed EFEMP1 expression was downregulated in lung cancer cells. The promoter region of EFEMP1 was methylated in A549 and H1299 and after treatment with 5-aza-2'-deoxycytidine, the EFEMP1 expression was upregulated. The growth and invasion of A549 and H1299 were all significantly suppressed by transfecting with EFEMP1 and the MMP-7 expression was dowanregulated by EFEMP1 as well. Expression activity of MMP-7 reporter construct was decreased by cotransfecting with EFEMP1.
Collectively, these results suggest that EFEMP1 functions as a suppressor of lung cancer growth and invasion. Epigenetic silencing of EFEMP1 promotes lung cancer invasion and metastasis by activating MMP-7 expression.
EFEMP1是纤连蛋白家族蛋白成员，是一种非常重要的细胞外基质蛋白，参与细胞代谢，但其在肿瘤发生和进展中的作用仍知之甚少。本研究旨在探讨EFEMP1在肺癌细胞生长和侵袭中的功能作用及机制。
通过蛋白质免疫印迹法检测肺癌细胞中EFEMP1的表达。采用甲基化特异性PCR（MSP）检测EFEMP1的启动子甲基化状态。在肺癌细胞中转染对照或EFEMP1载体后，通过集落形成实验和基质胶侵袭法检测集落形成和侵袭能力。采用蛋白质免疫印迹法和实时定量PCR检测基质金属蛋白酶-7（MMP-7）的表达。利用荧光素酶报告基因检测法检测在肺癌细胞中有无EFEMP1转染时MMP-7报告基因构建体的表达。
蛋白质免疫印迹结果显示肺癌细胞中EFEMP1表达下调。A549和H1299细胞中EFEMP1启动子区域发生甲基化，用5-氮杂-2'-脱氧胞苷处理后，EFEMP1表达上调。转染EFEMP1可显著抑制A549和H1299细胞的生长和侵袭，且EFEMP1也下调了MMP-7的表达。共转染EFEMP1可降低MMP-7报告基因构建体的表达活性。
总体而言，这些结果表明EFEMP1作为肺癌生长和侵袭的抑制因子发挥作用。EFEMP1的表观遗传沉默通过激活MMP-7表达促进肺癌侵袭和转移。
