A20诱导沉默对鼻咽癌细胞生物学行为的影响

Effect of A20 gene induced silencing on the biological behaviors of human nasopharyngeal carcinoma cell

目的观察A20基因表达下调对鼻咽癌细胞体内外增殖、侵袭及转移能力的影响。方法筛选并建立Tet－on基因表达系统诱导A20沉默的鼻咽癌肝转移亚系5－8F－H3细胞系，实时荧光定量PCR、Western印迹检测干扰效率；CCK8法、平板克隆形成实验检测A20对5－8F－H3增殖能力的影响；流式细胞仪检测细胞周期；侵袭小室检测A20对5－8F－H3侵袭能力的影响；裸鼠皮下成瘤实验和体内转移实验检测经强力霉素（DOX）诱导A20基因表达沉默对5－8F－H3在体内增殖及转移能力的影响。结果可诱导沉默A20的鼻咽癌细胞5－8F－H3/A20－shRNA构建成功。5－8F－H3/A20－shRNA经DOX诱导后，A20基因mRNA及蛋白的表达均下调（均P〈0.01）；CCK8法（F＝18.542，P＝0.003）、平板克隆形成实验（F＝40.080，P〈0.001）及流式细胞术检测实验（F＝7.398，P＝0.024）结果显示A20基因表达下调明显抑制5－8F－H3在体外增殖能力；侵袭小室检测结果显示A20基因表达下调抑制5－8F－H3在体外侵袭能力（F＝26.157，P〈0.001）。裸鼠皮下成瘤实验和体内转移实验结果显示A20基因表达下调明显抑制5－8F－H3在裸鼠体内的成瘤和转移能力。结论A20与鼻咽癌多种恶性生物学行为密切相关，有望成为鼻咽癌潜在治疗靶点。

Objective To observe the effect of knockdown A20 gene expression on the proliferation, invasion and metastasis of human nasopharyngeal carcinoma cell in vivo and in vivo.Methods Human nasopharyngeal carcinoma cell 5-8F-H3 was transfected with A20-specific shRNA Tet-on inducible plasmid vectors, and A20 silenced cells were screened by Puromycin. Quantitative RT-PCR and Western blot analysis were used to detect the mRNA level and protein of A20. The cell proliferation was detected by cell counting kit-8 （CCK8） and plate colony formation assays. The cell cycle and apoptosis were measured by flow cytometry. And the ability of cell invasion was measured using Boyden chamber assay in vivo. Subcutaneous tumor formation and liver metastasis in vivo were examined with whole-body fluorescence imaging system to observe the influence of silencing A20 gene expression in nude mice.Results The stable A20 inducible silencing cells line 5-8F-H3/A20-shRNA was established successfully. Down-regulation of A20 mRNA and protein expression were observed in 5-8F-H3/A20-shRNA cells treated with DOX（both P〈0.01）. The results of CCK-8 assay （F=18.542, P=0.003）, clone formation experiment （F=40.080, P〈0.001） and flow cytometry analysis （F=7.398, P=0.024） in vivo showed that the cell proliferation of 5-8F-H3 was remarkably inhibited by down-regulation of A20 gene expression. The results of Boyden chamber assay showed that A20 gene silencing could inhibit the cell invasion ability （F=26.157, P〈0.001）. Silencing of A20 inhibited tumorigenesis and metastasis via subcutaneous tumor formation and liver metastasis experiments in nude mice.Conclusion A20 gene is closely related to the malignant biological behaviors of nasopharyngeal carcinoma, and it may serve as a potential molecular target for the treatment of nasopharyngeal carcinoma.