Thy-1表达水平对肺腺癌细胞A549迁移性及部分相关基因表达的影响

目的 验证Thy-1(CD90)基因与肺癌细胞迁移潜能的关系,并探讨其作用机制. 方法 利用真核表达载体pcDNA3.0构建包含Thy-1基因全长序列的重组表达质粒载体,转染A549细胞,G418筛选获得稳定克隆,采用RT-PCR及免疫荧光技术检测重组质粒转染后细胞内Thy-1 mRNA及蛋白表达水平的改变;通过体外Transwell迁移实验研究外源性Thy-1对A549细胞迁移能力的影响. 结果 构建了pcDNA3.0-Thy-1真核细胞表达载体;成功建立了稳定表达外源性人Thy-1蛋白的肺癌A549细胞株.Trenswell实验提示,表达外源性Thy-1蛋白的A549细胞较对照组细胞体外迁移能力下降(P<0.0001);基因芯片结果提示,Thy-1基因在肺癌细胞中表达后,影响了25个细胞迁移相关基因的表达,在检测基因表达差异时,RT-PCR与基因芯片能够得到基本一致的结果. 结论 Thy-1基因的过表达能降低肺癌细胞系A549细胞的迁移能力,并改变A549细胞多个细胞迁移相关基因的表达,提示Thy-1在肺癌组织的表达状态可能影响肺癌的转移性.

The effects of Thy-1 expression on cell motility and regulated genes of lung adenocarcinama cell line A549 cells

Objective To investigate the function of Thy-1 gene, especially its possible roles in the migration of A549 cells as well as the possible mechanisms by which it works. Methods Construct recombinant expression plasmid vector of pcDNA3.0-Thy-1 and transfected it into A549 cells. These cell lines were further studied for the exogenous Thy-1 mRNA expression by RT-PCR and expression of endogenous Thy-1 protein by immunofluorescence assay. Then the transwell cell migrations were assayed. In order to study the possible mechanisms by which Thy-1 influences the invasiveness potential, microarray were used to screen out cell migrations associated genes. Results Eukaryotic expression vector pcDNA3.0-Thy-1 was constructed; Stable Thy-1 expressing cell line were established; Transwell assay showed that over expression of Thy-1 in A549 cell line resulted in reduced ability of cell migration (EM>P/EM><0.0001); and microarray suggested that the Thy-1 gene could influenced the expression level of 25 cell motility-related genes. In these genes， 11 genes expressions were up regulated, and 14 genes were down regulated. These results could be consistent when microarray and RT-PCR methods were used to detect the difference of gene expression.Conclusion Overexpression of Thy-1 leads to a less invasive phenotype of A549 cells and changes the expression level of many cell motility-related genes. This suggested that Thy-1 expression patterns in lung cancer tissue may be associated with the invasiveness of non-small cell lung cancer.