Zhao X F, Wang Q, Sun J, Zhang A M, Chang X Y, Li W G, Duan X Z
PLA Medical College, Beijing 100039, China Department of Oncology, Xi'an Daxing Hospital, Xi'an 710003.
Department of Radiation Oncology, The Fifth Medical Center of PLA General Hospital, Beijing 100039, China.
Zhonghua Gan Zang Bing Za Zhi. 2024 Sep 20;32(9):835-844. doi: 10.3760/cma.j.cn501113-20231130-00254.
To investigate the effect and associated mechanism of tumor tissue-infiltrating NK cells after receiving radiotherapy for hepatocellular carcinoma (HCC). A HCC tumor-bearing mouse model was constructed using human hepatocellular carcinoma cell line (SK-Hep-1) and divided into four groups: control, radiotherapy, NK cell clearance, and NK clearance combined with radiotherapy. Tumor growth condition was simultaneously recorded. The NK cell ratio in peripheral blood and the NK cell intratumoral infiltration condition were detected by flow cytometry and immunohistochemistry. Lentiviral-constructed SK-Hep-1 cells was used to detect the effect of radiotherapy on the regulation of CXCL10 and NK cell chemotaxis following EZH2 overexpression. SK-Hep-1 cells were irradiated and . The expression levels of EZH2 and CXCL10 mRNA and protein in the two groups of cell lines and mouse tumor tissues were detected by reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), western blotting (WB), and immunohistochemistry. The chemotaxis and blocking experiments were used to validate the chemotaxis effect of CXCL10 on NK cells. The independent sample t-test was used to compare the groups. <0.05 was considered statistically significant. The HCC tumor-bearing mouse model experiment showed that HCC tumor growth was most remarkable in the NK clearance combined with the radiotherapy group compared to the radiotherapy group (<0.05). Compared with the control group, the number of NK cells in the peripheral blood of nude mice in the radiotherapy group was significantly reduced, while the NK cell intratumoral infiltration was significantly increased (<0.05). Flow cytometry and immunohistochemistry showed and expressional alterations. The average expression levels of EZH2 mRNA and protein in hepatocellular carcinoma cell lines and tumor tissues were decreased in the radiotherapy group than the control group and mouse tumor tissues (<0.05), while the mRNA and protein expression levels of CXCL10 increased (<0.05). The cell supernatant following radiotherapy enhanced NK cell chemotaxis but inhibited CXCL10 neutralization. EZH2 overexpression validated that radiotherapy up-regulated CXCL10 mRNA and down-regulated protein expression levels in in vitro and in vivo experiments (<0.05). The chemotactic effect on NK cells was significantly weakened with EZH2 overexpression following radiotherapy. NK cells, as immune effector cells, are directly involved in radiotherapy- activated anti-HCC immunity. Importantly, radiotherapy inhibits EZH2 expression in hepatocellular carcinoma, thereby upregulating CXCL10 expression and enhancing intratumoral NK cell invasion.
研究肝细胞癌（HCC）放疗后肿瘤组织浸润性NK细胞的作用及相关机制。用人肝癌细胞系（SK-Hep-1）构建荷HCC肿瘤小鼠模型，并分为四组：对照组、放疗组、NK细胞清除组、NK细胞清除联合放疗组。同时记录肿瘤生长情况。采用流式细胞术和免疫组织化学检测外周血NK细胞比例及肿瘤内NK细胞浸润情况。利用慢病毒构建的SK-Hep-1细胞检测放疗对EZH2过表达后CXCL10调控及NK细胞趋化性的影响。对SK-Hep-1细胞进行照射。通过逆转录聚合酶链反应（RT-PCR）、酶联免疫吸附测定（ELISA）、蛋白质印迹法（WB）和免疫组织化学检测两组细胞系及小鼠肿瘤组织中EZH2和CXCL10 mRNA及蛋白的表达水平。采用趋化性和阻断实验验证CXCL10对NK细胞的趋化作用。采用独立样本t检验比较各组。P<0.05认为差异有统计学意义。荷HCC肿瘤小鼠模型实验显示，与放疗组相比，NK细胞清除联合放疗组HCC肿瘤生长最为显著（P<0.05）。与对照组相比，放疗组裸鼠外周血NK细胞数量显著减少，而肿瘤内NK细胞浸润显著增加（P<0.05）。流式细胞术和免疫组织化学显示表达改变。放疗组肝癌细胞系和肿瘤组织中EZH2 mRNA和蛋白的平均表达水平低于对照组和小鼠肿瘤组织（P<0.05），而CXCL10的mRNA和蛋白表达水平升高（P<0.05）。放疗后的细胞上清液增强了NK细胞趋化性，但抑制了CXCL10中和作用。EZH2过表达验证了放疗在体外和体内实验中上调CXCL10 mRNA并下调蛋白表达水平（P<0.05）。放疗后EZH2过表达使对NK细胞的趋化作用显著减弱。NK细胞作为免疫效应细胞，直接参与放疗激活的抗HCC免疫。重要的是，放疗抑制肝癌中EZH2表达，从而上调CXCL10表达并增强肿瘤内NK细胞浸润。
