FOXG1在结直肠癌侵袭及转移中的作用及机制

Role and mechanism of FOXG1 in invasion and metastasis of colorectal cancer

摘要:

构建叉头框G1 (Forkhead box G1, FOXG1) 的慢病毒干扰 (shRNA) 质粒及表达质粒，通过敲低和过表达FOXG1探讨其对结直肠癌细胞上皮-间质转化EMT的作用及其机制。应用Western blotting检测FOXG1在RKO、SW480、SW620、LOVO、DLD-1五种结直肠癌细胞中蛋白的表达水平，设计并合成FOXG1的shRNA片段 (shFOXG1)，运用DNA重组技术获得重组质粒，经双酶切技术及测序方法鉴定后进行慢病毒的包装、纯化及稳定转染，经筛选后获得稳定的结直肠癌细胞株，通过Western blotting和qRT-PCR技术检测FOXG1敲低和过表达效率及EMT关键因子E-cadherin、Vimentin、Fibronectin、Snail、Twist mRNA和蛋白的变化，光学显微镜观察敲低后细胞形态学变化，通过划痕实验检测迁移能力变化，Transwell检测侵袭迁移能力的变化。5种结直肠癌细胞中，FOXG1在RKO细胞中蛋白表达量最高，而在DLD-1细胞中表达量最低，与对照组相比较，在RKO细胞中敲低FOXG1，细胞形态由长梭型变成了类圆形或者多边形，细胞极性和紧密连接增加，细胞迁移距离明显降低，侵袭转移穿过小室的细胞数也明显减少，EMT关键因子E-cadherin表达增高，Vimentin、Fibronectin、Snail、Twist表达降低，过表达FOXG1组则相反。FOXG1在结直肠癌中高表达，这种基因的高表达能够促进结直肠癌细胞的侵袭和转移，对结直肠癌细胞的EMT起着重要的调控作用。

Abstract:

This study was aimed to investigate the effect of Forkhead Box G1 (FOXG1) on the epithelial-mesenchymal transition (EMT) of colorectal cancer (CRC) cells and the underlying mechanism. For this purpose, FOXG1 lentiviral interference (shRNA) plasmid and expression plasmid were constructed. Western blotting was used to analyze the expression of FOXG1 protein in five CRC cells, namely RKO, SW480, SW620, LoVo and DLD-1. The shRNA fragment of FOXG1 (shFOXG1) was designed and synthesized. Recombinant plasmids were obtained with the aid of DNA recombination technique. Double digestion and sequencing were used to identify the recombinant plasmids, and then lentivirus packaging, purification and stable transfection were carried out. Additionally, stable CRC cell lines were screened out. The changes of FOXG1 knockdown and overexpression efficiency, E-cadherin, Vimentin, Fibronectin, Snail, Twist mRNA and protein were investigated respectively by Western blotting and qRT-PCR analysis. Furthermore, the changes of cell morphology after knockdown and cell migration ability were evaluated respectively with optical microscopy, scratch test and Transwell assay. FOXG1 had the highest protein expression in RKO and the lowest in DLD-1 among the five CRC cells. Compared with those of the control group, the cell morphology in FOXG1 knockdown RKO group was changed from spindle into round or polygonal shape, cell polarization was enhanced and tight junction assembly was acclerated while cell migration distance was noticeably decreased. Moreover, the number of cells invaded and migrated through chambers was significantly reduced. Among these key factors of EMT, the expression of E-cadherin was increased while the expressions of Vimentin, Fibronectin, Snail and Twist were decreased. The opposite was the case in the overexpressed FOXG1 group. The overexpression of FOXG1 in CRC promoted the invasion and metastasis of CRC cells and played a crucial role in regulating the EMT. Thus, FOXG1 might be a novel therapeutic target in CRC treatment.