沉默HIG2促进NK细胞对喉癌细胞杀伤敏感性的机制探讨

The mechanism of silencing HIG2 promoting the killing sensitivity of NK cells to laryngeal carcinoma cells

目的:探讨缺氧诱导基因2(HIG2)在喉癌细胞逃逸NK细胞免疫杀伤中的作用机制。方法:免疫组织化学法检测喉癌组织和癌旁组织中HIG2的表达;Western blot检测HIG2、NKG2D配体(MICA、MICB、ULBP1、ULBP2和ULBP3)的蛋白表达;Transwell实验检测Hep-2细胞的侵袭;流式细胞术检测Hep-2细胞的凋亡、CD3^(-)CD56^(+)的NK细胞纯度、NKG2D受体表达及Hep-2细胞NKG2D配体的表达;乳酸脱氢酶释放法检测不同效靶比时,NK细胞对Hep-2细胞的杀伤活性。结果:HIG2在喉癌组织中高表达。CD3^(-)CD56^(+)的NK细胞纯度大于90%。沉默HIG2抑制Hep-2细胞侵袭,促进细胞凋亡,提高NKG2D配体的表达,增强NK细胞对Hep-2细胞的杀伤敏感性。NKG2D抗体抑制NK细胞NKG2D受体表达,减弱NK细胞对沉默HIG2的Hep-2细胞的杀伤活性。结论:沉默HIG2可抑制喉癌细胞侵袭,促进细胞凋亡,上调NKG2D配体表达,增强NK细胞对喉癌细胞的杀伤敏感性。

Objective:To investigate the mechanism of HIG2-mediated immune escape of laryngeal cancer cells from NK cell immune killing.Methods:Immunohistochemistry was used to detect the expression of HIG2 in laryngeal cancer tissues and adjacent tissues.Western blot was used to detect the protein level of HIG2 and NKG2D ligands(MICA,MICB,ULBP1,ULBP2 and ULBP3).Transwell was used to detect invasion of Hep-2 cells.Flow cytometry was used to detect Hep-2 cell apoptosis,CD3^(-)CD56^(+)NK cell purity and NKG2D receptor expression,and expression of Hep-2 cell NKG2D ligand.LDH releasing assay was used to detect cytotoxicity of NK cells against Hep-2 cells at effect-to-target cell ratios of 5∶1,10∶1,20∶1.Results:The expression level of HIG2 was up-regulated in laryngeal cancer tissues.The purity of CD3^(-)CD56^(+)NK cells is greater than 90%.Silencing HIG2 inhibited Hep-2 cell invasion,promoted apoptosis,increased the expression of NKG2D ligands(MICA,MICB,ULBP1,ULBP2,and ULBP3),and enhanced the cytotoxicity of NK cells against Hep-2 cells.The NKG2D monoclonal antibody inhibited the expression of NKG2D receptors on NK cells and attenuates the cytotoxicity of NK cells against Hep-2 cells that silence HIG2.Conclusion:Silencing HIG2 inhibited laryngeal carcinoma cell invasion,promoted apoptosis,enhanced the killing sensitivity of NK cells to laryngeal carcinoma cells by up-regulating the expression of NKG2D ligands.