癌基因Src在骨肉瘤细胞侵袭伪足形成中的作用

Effects of Oncogene Src on Invadopodia Formation of Osteosarcoma Cells

目的:探讨癌基因Src在体外培养骨肉瘤细胞侵袭伪足形成中的作用。方法:构建Src sh RNA慢病毒表达载体,在HEK293T细胞中包装慢病毒,感染HT-1080骨肉瘤细胞,经嘌呤霉素加压筛选,获得稳定沉默Src基因的骨肉瘤细胞系HT-1080-sh Src;实时定量PCR和Western Blot法检测基因沉默效率;采用原位明胶酶谱法检测侵袭伪足形成;采用侵袭小室实验检测下调Src基因表达对HT-1080细胞侵袭力的影响。结果:成功构建稳定沉默Src基因的骨肉瘤细胞系HT-1080-sh Src及对照细胞系HT-1080-shluc,经实时定量PCR和Western Blot检测,与对照细胞系相比,HT-1080-sh Src细胞中Src基因表达下调3倍以上;下调HT-1080细胞中Src基因表达能显著抑制HT-1080细胞侵袭伪足形成及其对细胞外基质的降解能力;下调Src基因表达能显著抑制骨肉瘤细胞侵袭力。结论:癌基因Src参与调节骨肉瘤细胞HT-1080侵袭伪足形成,促进肿瘤侵袭、转移。

Objective： To investigate the effects of oncogene Src on invadopodia formation of osteosarcoma cells （HT-1080） in vitro. Methods： Src shRNA lentiviral vector was constructed, packaged in HEK293T cells. And HT-1080 cells were infected, then screened by puromycin. And then stablely Src gene silencing osteosarcoma cell line HT-1080-shSrc was obtained. Quantification PCR and Western Blot were carried out to detect the gene silencing efficiency. Invadopodia formation was observed by in situ gelatin zymography. In vitro invasion assays were used to detect the effects of downregulation Sre expression on invasive ability of HT-1080 cells. Results： Stablely Src gene silencing osteosarcoma cell line HT-1080-shSrc and control cell line HT-1080-shluc were successfully constructed. Quantification PCR and Western Blot showed, compared with HT-1080-shluc cells, the expression of Src in HT-1080-shSrc cells decreased more than 3 times. Invadopodia formation, the ability to degradate the extracellular matrix and the invasive ability were significantly inhibited when Src expression was downregulated in HT-1080 cells. Conclusion： Oncogene Src is involved in regulating invadopodia formation in osteosarcoma cells HT-1080, and promoting tumor invasion and metastasis.