Objective To investigate the inhibition on the proliferation and metastasis of laryngeal squamous carcinoma Hep-2 cells by the expression of Syk (L) gene through the demethylation of the full length spleen tyrosine kinase [Syk (L)] gene.Methods The proliferation of Hep-2 cell which was treated with different concentrations of Trichosanthin at different time points was tested by CCK-8 assay,and the ability of invasion and metastasis of Hep-2 cells treated after 72 hours was detected by Transwell assay.Methyla tion level and the expression of Syk (L) gene in Hep-2 cells which were treated with different concentrations of Trichosanthin were detected through MS-HRM,Q-RT-PCR and Western blot.Results The results of CCK-8 showed that Trichosanthin inhibited the proliferation of Hep-2 cells with the dependence of concentration and time in a dose-and time-dependent manner.Transwell results showed that Trichosanthin significantly inhibited the invasion and metastasis of Hep-2 cells.MS-HRM showed that Trichosanthin could reverse the hypermethylation of the Sky (L) gene in Hep-2 cells and restore the expression of Syk (L) mRNA and protein.Conclusion Trichosanthin activates Syk (L) gene expression in Hep-2 cells and restores Syk mRNA and protein expression by reversing the hypermethylation status of CpG island of Syk (L) gene promoter,thus exerts anti-oncogene effection to inhibit the malignant biological behavior of Hep-2 cells,which has potential values in being developed into antitumor drugs.%目的 探讨天花粉蛋白通过全长型脾络氨酸激酶[full-length form of spleen tyrosine kinase,Syk(L)]基因去甲基化途径恢复Syk(L)表达对喉癌Hep-2细胞增殖、侵袭迁移的的抑制作用.方法 采用CCK-8法及Transwell实验检测不同浓度天花粉在不同时间点对Hep-2细胞增殖及处理72h后细胞侵袭迁移能力的影响;采用MS-HRM法、Q-RT-PCR及Westernblot法检测不同浓度天花粉蛋白对Hep-2细胞Syk(L)基因甲基化水平及其表达的影响.结果 CCK-8结果显示,天花粉蛋白对喉癌Hep-2细胞的增殖有抑制作用,且呈浓度、结果时间依赖性;Transwell结果显示,天花粉蛋白能显著抑制Hep-2细胞的侵袭迁移能力;MS-HRM结果显示,天花粉蛋白可以逆转Hep-2细胞Sky(L)基因的高甲基化状态,恢复Syk(L) mRNA及蛋白的表达.结论 天花粉蛋白通过逆转Syk(L)基因启动子CpG岛的甲基化状态,使喉癌Hep-2细胞中Syk(L)基因表达活化,恢复Syk mRNA及蛋白的表达,最终发挥抑癌基因的作用抑制Hep-2细胞恶性生物学行为,具有开发并成为抗肿瘤药物的潜在价值.
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