天花粉蛋白诱导Syk(L)基因去甲基化抑制喉癌Hep-2细胞生长研究

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Western blot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。

Research on the Demethylation of Sky(L) Gene Induced by Trichosanthin to Inhibit the Growth of Laryngeal Carcinoma Hep-2 Cell

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. Methylation 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.