GPC5基因在肺腺癌细胞系中的功能研究

Functions of glypican-5 gene in lung adenocarcinoma cell lines

目的研究GPC5基因在肺腺癌细胞系中的表达情况及其对肺腺癌细胞系生物学行为的影响。方法采用逆转录PCR(RT-PCR)、实时定量PCR(qRT-PCR)以及Western blot分析肺腺癌细胞系中GPC5基因mRNA及蛋白表达情况;构建GPC5过表达载体及干扰载体,通过转染构建GPC5过表达细胞系及GPC5沉默细胞系;利用CCK-8法分析肺腺癌细胞系生长情况;通过流式细胞术检测肺腺癌细胞系的周期及凋亡变化。结果检测了GPC5基因在4株肺腺癌细胞系(A549、H1299、H358和H1975)和1株肺正常上皮细胞系(HBE)中的mRNA表达情况,发现与HBE细胞系相比,GPC5基因在A549细胞系中呈低表达,在H1299细胞系中高表达,同时蛋白检测结果与mRNA水平一致;A549细胞中过表达GPC5基因可抑制细胞生长,引起细胞周期阻滞;在H1299细胞中干扰GPC5基因表达促进细胞生长,促进细胞周期;在过表达和干扰细胞模型中均未发现GPC5基因对细胞凋亡的显著影响。结论 GPC5基因可以对肺腺癌细胞系的生物学行为产生影响,可能是肺腺癌的抑癌基因。

Objective To determine the expression pattern of glypican-5 （GPC5） gene in the lung adenocarcinoma cell lines and the effects of its over-expression or silencing on the biological behaviors in lung adenocarcinoma cell lines. Methods The expression of GPC5 gene at mRNA and the protein levels in 4 lung adenocarcinoma cell lines （A549, H1299, H358 and H1975） and 1 strain of normal lung epithelial cell line （HBE） was determined by reverse transcription PCR （RT-PCR）, real-time quantitative PCR （qRT- PCR） and Western blotting. Over-expression vectors of GPC5 gene and its interference plasmids were constructed, and then transfected respectively into A549 cells （lower expression of the gene） or H1299 cells （higher expression） to over-express or silence the gene. Cell counting kit-8 （CCK-8） assay was performed to measure the proliferation of the lung adenocarcinoma cell lines. Cell cycle and apoptosis were detected by flow cytometry. Results Compared with the expression level of GPC5 in normal cells （ HBE）, the level was lower in A549 cells and highly in H1299 cells, with its protein level consistent with the mRNA level. Over- expression of GPC5 in A549 cells gene resulted in inhibition of cell growth and arrest of cells at G_1/S phase.On the other hand, interference of GPC5 in H1299 cells promoted cell growth and improved cell cycle. Over- expression and interference of GPC5 gene significantly affected the cell apoptosis in the lung adenocarcinoma cell lines. Conclusion GPC5 gene affects the biological behaviors of lung adenocarcinoma cell lines. It may be a tumor suppressor gene, and its specific molecular mechanism is worth of further research.