Qin Jin, Liu Zheng-Xiang
Department of Cardiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, P. R. China.
Ai Zheng. 2006 Jul;25(7):833-8.
BACKGROUND & OBJECTIVE: Over-expression or over-activation of focal adhesion kinase (FAK) correlates with cancer migration. FAK related non-kinase (FRNK) acts as an endogenous inhibitor of FAK, which can compete with FAK for focal adhesion binding sites. This study was designed to determine the inhibitory effects of FRNK gene on migration of a human breast carcinoma cell line MDA-MB-435 and explore the possible mechanisms.
The functional fragment of FRNK cDNA was amplified by reverse transcription polymerase chain reaction (RT-PCR) and cloned into pcDNA3.1 vector. The recombinant pcDNA3.1-FRNK was transfected into MDA-MB-435 cells using lipofectamine 2000. The stably transfected cells were selected in a medium containing geneticin (G418). Expression of FRNK in stably transfected MDA-MB-435 cells and MMP-9 in both wild-type and transfected MDA-MB-435 cells was detected by Western blot. The effect of FRNK on cell migration was determined using cell wound healing and Boyden chamber assays, respectively, in vitro.
The recombinant plasmid pcDNA3.1-FRNK was successfully constructed and MDA-MB-435 cells stably transfected with pcDNA3.1-FRNK were obtained. MMP-9 protein expression was inhibited by 73.1% in MDA-MB-435 cells transfected with pcDNA3.1-FRNK compared to wild-type cells. In wound healing study, migrated cell count was significantly lower in MDA-MB-435/FRNK cells (0.35+/-0.02) than that in wild type cells (0.58+/-0.06, P<0.05). In Boyden chamber assay, the number of migrated MDA-MB-435/FRNK cells was (65.15+/-8.56), which was 66.57% of migrated wild type cells (97.86+/-5.44).
These findings suggest that FRNK may inhibit the migration of the human breast carcinoma cell line MDA-MB-435. And the suppressive effect may be due to the down-regulation of MMP-9 in MDA-MB-435 cells.
粘着斑激酶（FAK）的过表达或过度激活与癌症迁移相关。FAK相关非激酶（FRNK）作为FAK的内源性抑制剂，可与FAK竞争粘着斑结合位点。本研究旨在确定FRNK基因对人乳腺癌细胞系MDA-MB-435迁移的抑制作用，并探讨其可能机制。
通过逆转录聚合酶链反应（RT-PCR）扩增FRNK cDNA的功能片段，并将其克隆到pcDNA3.1载体中。使用脂质体2000将重组pcDNA3.1-FRNK转染到MDA-MB-435细胞中。在含有遗传霉素（G418）的培养基中筛选稳定转染的细胞。通过蛋白质免疫印迹法检测稳定转染的MDA-MB-435细胞中FRNK的表达以及野生型和转染的MDA-MB-435细胞中基质金属蛋白酶-9（MMP-9）的表达。分别使用细胞划痕愈合实验和Boyden小室实验在体外确定FRNK对细胞迁移的影响。
成功构建了重组质粒pcDNA3.1-FRNK，并获得了稳定转染pcDNA3.1-FRNK的MDA-MB-435细胞。与野生型细胞相比，转染pcDNA3.1-FRNK的MDA-MB-435细胞中MMP-9蛋白表达受到73.1%的抑制。在划痕愈合实验中，MDA-MB-435/FRNK细胞的迁移细胞数（0.35±0.02）明显低于野生型细胞（其迁移细胞数为0.58±0.06，P<0.05）。在Boyden小室实验中，MDA-MB-435/FRNK细胞的迁移数为（65.15±8.56），是野生型细胞迁移数（97.86±5.44）的66.57%。
这些发现表明FRNK可能抑制人乳腺癌细胞系MDA-MB-435的迁移。并且这种抑制作用可能是由于MDA-MB-435细胞中MMP-9表达下调所致。
