LncRNA HCG18影响鼻咽癌细胞PD-L1表达及CD8<sup>+</sup>T细胞的杀伤功能研究

doi:10.3969/j.issn.1006-6233.2024.07.03

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摘要 目的: 探究LncRNA HCG18调控CD8⁺T细胞抗鼻咽癌细胞的功能及机制。方法: qRT-PCR检测鼻咽癌组织及癌旁组织HCG18表达以及人鼻黏膜上皮细胞HNEpC和人鼻咽癌细胞系CNE1、CNE2、HONE-1、HNE-2中HCG18表达。CNE1细胞分为si-HCG18组、si-NC组、si-HCG18+PD-L1组,HNE-2细胞分为HCG18组、Vector组和HCG18+sh-PD-L1组,上述各组CNE1细胞和HNE-2细胞分别与CD8⁺T细胞共孵育24h(si-HCG18+CD8⁺T组、si-NC+CD8⁺T组、si-HCG18+PD-L1+CD8⁺T组、HCG18+CD8⁺T组、Vector+CD8⁺T组和HCG18+sh-PD-L1+CD8⁺T组),酶联免疫吸附试验(Enzyme Linked Immunosorbent Assay,ELISA)检测共孵育体系中INF-γ、TNF-α、IL-2浓度,细胞毒性试剂盒检测肿瘤细胞裂解率。Western blot检测PD-L1蛋白表达。双荧光素酶报告基因实验验证HCG18与miR-20b-5p的靶向关系,miR-20b-5p与PD-L1的靶向关系。结果: 鼻咽癌组织中HCG18表达显著高于癌旁组织,CNE1、CNE2、HONE-1、HNE-2细胞中HCG18表达均显著高于HNEpC细胞(P<0.05)。si-HCG18组CNE1细胞中PD-L1表达显著低于si-NC组(P<0.05),HCG18组HNE-2细胞PD-L1表达显著高于Vector组(P<0.05),相比于si-NC+CD8⁺T组,si-HCG18+CD8⁺T组共孵育体系中INF-γ、TNF-α、IL-2浓度显著增加(P<0.05),CNE1细胞裂解率显著增加(P<0.05),相比于si-HCG18+CD8⁺T组,si-HCG18+PD-L1+CD8⁺T组共孵育体系中INF-γ、TNF-α、IL-2浓度显著降低(P<0.05),CNE1细胞裂解率显著降低(P<0.05)。相比于Vector+CD8⁺T组,HCG18+CD8⁺T组共孵育体系中INF-γ、TNF-α、IL-2浓度显著降低(P<0.05),HNE-2细胞裂解率显著降低(P<0.05),相比于HCG18+CD8⁺T组,HCG18+sh-PD-L1+CD8⁺T组共孵育体系中INF-γ、TNF-α、IL-2浓度显著增加(P<0.05),HNE-2细胞裂解率显著增加(P<0.05)。miR-20b-5p为HCG18靶基因,PD-L1为miR-20b-5p靶基因。结论: HCG18上调鼻咽癌细胞PD-L1表达并且抑制CD8⁺T细胞的抗肿瘤活性。

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关键词 ：鼻咽癌, HCG18, PD-L1, CD8⁺T细胞

Abstract：Objective: To investigate the regulatory mechanism of LncRNA HCG18 on the function of CD8⁺ T cells against nasopharyngeal carcinoma (NPC) cells. Methods: The expression of HCG18 in NPC tissues and paracancerous tissues was detected by qRT-PCR. The expression of HCG18 in human nasal mucosal epithelial cells (HNEpC) and NPC cell lines (CNE1, CNE2, HONE-1, HNE-2) was also detected. CNE1 cells were divided into si-HCG18 group, si-NC group, and si-HCG18⁺PD-L1 group; HNE-2 cells were divided into HCG18 group, Vector group, and HCG18⁺sh-PD-L1 group. CNE1 and HNE-2 cells in each group were co-cultured with CD8⁺ T cells for 24 h (si-HCG18+CD8⁺T group, si-NC+CD8⁺T group, si-HCG18+PD-L1+CD8⁺T group, HCG18+CD8⁺T group, Vector+CD8⁺T group, and HCG18+sh-PD-L1+CD8⁺T group). The concentrations of INF-γ, TNF-α, and IL-2 in the co-culture system were detected by enzyme-linked immunosorbent assay (ELISA), and the tumor cell lysis rate was detected by a cytotoxicity assay kit. Western blot was used to detect PD-L1 protein expression. Dual luciferase reporter gene assay was used to verify the targeting relationship between HCG18 and miR-20b-5p, and the targeting relationship between miR-20b-5p and PD-L1. Results: The expression of HCG18 in NPC tissues was significantly higher than that in paracancerous tissues, and the expression of HCG18 in CNE1, CNE2, HONE-1, and HNE-2 cells was significantly higher than that in HNEpC cells (P<0.05). The expression of PD-L1 in CNE1 cells of the si-HCG18 group was significantly lower than that in the si-NC group (P<0.05), and the expression of PD-L1 in HNE-2 cells of the HCG18 group was significantly higher than that in the Vector group (P<0.05). Compared with the si-NC+CD8⁺T group, the concentrations of INF-γ, TNF-α, and IL-2 in the co-culture system of the si-HCG18+CD8⁺T group were significantly increased (P<0.05), and the CNE1 cell lysis rate was significantly increased (P<0.05). Compared with the si-HCG18+CD8⁺T group, the concentrations of INF-γ, TNF-α, and IL-2 in the co-culture system of the si-HCG18+PD-L1+CD8⁺T group were significantly decreased (P<0.05), and the CNE1 cell lysis rate was significantly decreased (P<0.05). Compared with the Vector+CD8⁺T group, the concentrations of INF-γ, TNF-α, and IL-2 in the co-culture system of the HCG18+CD8⁺T group were significantly decreased (P<0.05), and the HNE-2 cell lysis rate was significantly decreased (P<0.05). Compared with the HCG18+CD8⁺T group, the concentrations of INF-γ, TNF-α, and IL-2 in the co-culture system of the HCG18+sh-PD-L1+CD8⁺T group were significantly increased (P<0.05), and the HNE-2 cell lysis rate was significantly increased (P<0.05). miR-20b-5p is a target gene of HCG18, and PD-L1 is a target gene of miR-20b-5p. Conclusion: HCG18 upregulates PD-L1 expression in NPC cells and suppresses the anti-tumor activity of CD8⁺ T cells.

Key words： Nasopharyngeal carcinoma HCG18 PD-L1 CD8+T cells

基金资助:新疆维吾尔自治区自然科学基金项目,(编号:2022D01C504)

通讯作者: 高妍

引用本文:

刘春江, 吴天宇, 高妍. LncRNA HCG18影响鼻咽癌细胞PD-L1表达及CD8⁺T细胞的杀伤功能研究[J]. 河北医学, 2024, 30(7): 1068-1074.
LIU Chunjiang, et al. Effect of LncRNA HCG18 on PD-L1 Expression in Nasopharyngeal Carcinoma Cells and CD8⁺ T Cell Cytotoxic Activity. HeBei Med, 2024, 30(7): 1068-1074.

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http://www.hbyxzzs.cn/CN/10.3969/j.issn.1006-6233.2024.07.03 或 http://www.hbyxzzs.cn/CN/Y2024/V30/I7/1068

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