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

Abstract

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.