国际肿瘤学杂志

国际肿瘤学杂志 ›› 2023, Vol. 50 ›› Issue (7): 407-412.doi: 10.3760/cma.j.cn371439-20230404-00079

• 论著 • 上一篇    下一篇

RNF43通过β-catenin抑制黑色素瘤细胞PD-L1表达并促进CD8+ T细胞介导的抗肿瘤免疫反应

吴旻杭1, 孙文政2, 于庆卓2, 郭蓉2, 叶辉3, 杜莹3, 邱晋2, 安华章2(), 曹莉莉1,2,3()   

  1. 1山东中医药大学第二临床医学院千佛山医院肿瘤中心，济南 250014
    2山东第一医科大学第一附属医院（山东省千佛山医院）肿瘤中心 山东省临床免疫转化医学高校实验室 山东省风湿免疫病转化医学重点实验室 山东省肺癌研究所，济南 250014
    3山东大学山东省千佛山医院肿瘤中心，济南 250014
  • 收稿日期:2023-04-04 修回日期:2023-06-03 出版日期:2023-07-08 发布日期:2023-08-03
  • 通讯作者: 曹莉莉，Email： cll@sdu.edu.cn；安华章，Email： anhz@immunol.org

RNF43 inhibits PD-L1 expression via β-catenin in melanoma cells and promotes CD8+ T cell-mediated anti-tumor immune reaction

Wu Minhang1, Sun Wenzheng2, Yu Qingzhuo2, Guo Rong2, Ye Hui3, Du Ying3, Qiu Jin2, An Huazhang2(), Cao Lili1,2,3()   

  1. 1Oncology Center，Qianfoshan Hospital，The Second Clinical College of Shandong University of Traditional Chinese Medicine，Jinan 250014，China
    2Oncology Center，The First Affiliated Hospital of Shandong First Medical University & Shandong Provincial Qianfoshan Hospital，Shandong Provincial University Laboratory of Clinical Immunology and Translational Medicine，Shandong Provincial Key Laboratory for Rheumatic Disease and Translational Medicine，Shandong Lung Cancer Institute, Jinan 250014, China
    3Oncology Center，Shandong Provincial Qianfoshan Hospital，School of Medicine，Shandong University，Jinan 250014，China
  • Received:2023-04-04 Revised:2023-06-03 Online:2023-07-08 Published:2023-08-03
  • Contact: Cao Lili，Email： cll@sdu.edu.cn；An Huazhang，Email： anhz@immunol.org

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摘要：

目的 探讨环指蛋白43（RNF43）对黑色素瘤中CD8+ T细胞介导的抗肿瘤免疫反应的调节作用。方法 利用慢病毒感染技术对小鼠黑色素瘤细胞株B16-OVA进行RNF43基因过表达与敲低；通过小鼠皮下成瘤实验检测Lv-Ctrl-OE组、Lv-RNF43-OE组、Lv-Ctrl-KD组和Lv-RNF43-KD组小鼠黑色素瘤细胞株B16-OVA体内增殖情况，通过流式细胞术检测黑色素瘤肿瘤免疫微环境中CD8+ T细胞穿孔素和γ干扰素（IFN-γ）的表达水平；采用实时荧光定量PCR实验检测细胞中β-catenin和程序性死亡受体配体1（PD-L1） mRNA的表达水平；通过双荧光素酶报告基因实验检测RNF43对PD-L1的转录调控作用。结果 本研究成功构建RNF43稳定过表达与敲低的小鼠黑色素瘤细胞株Lv-RNF43-OE和Lv-RNF43-KD。小鼠皮下成瘤实验结果显示，Lv-RNF43-OE组瘤体质量为（0.08±0.06）g，明显小于Lv-Ctrl-OE组的（1.04±0.52）g，差异有统计学意义（t=3.71，P=0.032）；Lv-RNF43-KD组瘤体质量为（1.94±0.29）g，与Lv-Ctrl-KD组的（1.15±0.74）g相比差异无统计学意义（t=-1.70，P=0.164）。流式细胞术结果显示，Lv-RNF43-OE组CD8+ T细胞穿孔素荧光强度为9 034±2 628，明显高于Lv-Ctrl-OE组的3 847 ±1 637，差异有统计学意义（t=-3.35，P=0.015）；Lv-RNF43-KD组CD8+ T细胞穿孔素荧光强度为966±247，明显低于Lv-Ctrl-KD组的2 226±646，差异有统计学意义（t=3.16，P=0.034）；Lv-RNF43-OE组CD8+ T细胞IFN-γ荧光强度为2 422±429，明显高于Lv-Ctrl-OE组的1 688±324，差异有统计学意义（t=-2.73，P=0.034）；Lv-RNF43-KD组CD8+T细胞IFN-γ荧光强度为614（454，863），与Lv-Ctrl-KD组的1 159（1 152，2 068）相比差异有统计学意义（Z=-1.96，P=0.050）。实时荧光定量PCR实验结果显示，Lv-RNF43-OE组β-catenin mRNA相对表达量为0.67±0.16，明显低于Lv-Ctrl-OE组的1.00±0.11，差异有统计学意义（t=2.98，P=0.041）；Lv-RNF43-OE组PD-L1 mRNA相对表达量为0.32±0.09，明显低于Lv-Ctrl-OE组的1.00±0.09，差异有统计学意义（t=9.13，P=0.001）。双荧光素酶报告基因实验结果显示，pCMV6-NC组、RNF43组、RNF43+β-catenin组和β-catenin组PD-L1启动子荧光素酶活性分别为1.00±0.00、0.84±0.00、1.49±0.00和1.57±0.03（F=2 218.33，P<0.001），进一步两两比较发现，与pCMV6-NC组相比，RNF43组PD-L1启动子荧光素酶活性明显降低（P<0.001），RNF43+β-catenin组和β-catenin组PD-L1启动子荧光素酶活性明显升高（P<0.001；P=0.003）；与RNF43组相比，RNF43+β-catenin组PD-L1启动子荧光素酶活性明显升高（P<0.001）。结论 RNF43可能通过抑制β-catenin的表达降低黑色素瘤中PD-L1 mRNA的表达并促进CD8+ T细胞介导的抗肿瘤免疫反应。

关键词: 黑色素瘤, β连环素, 环指蛋白43

Abstract:

Objective To investigate the regulatory effects of ring finger protein 43 （RNF43） on CD8+ T cell-mediated anti-tumor immune reaction in melanoma. Methods RNF43 gene was over-expressed and knockdown in mouse melanoma cells line B16-OVA by lentivirus infection； In vivo proliferation of mouse melanoma cells line B16-OVA in the Lv-Ctrl-OE，Lv-RNF43-OE，Lv-Ctrl-KD and Lv-RNF43-KD groups was detected by subcutaneous tumorigenesis assay in mice，and the expression levels of CD8+ T cells perforin and interferon γ （IFN-γ） in tumor immune microenvironment of melanoma were detected by flow cytometry； The expression levels of β-catenin and programmed death-ligand 1 （PD-L1） mRNA in cells were detected by quantitative real-time PCR assay； The effect of RNF43 on the transcriptional regulation of PD-L1 was detected by dual-luciferase reporter gene assay. Results Stable RNF43 over-expressing and RNF43 knockdown mouse melanoma cells lines Lv-RNF43-OE and Lv-RNF43-KD were successfully constructed. The results of subcutaneous tumorigenesis experiment in mice showed that the tumor mass of the Lv-RNF43-OE group was （0.08±0.06） g，which was significantly smaller than that of the Lv-Ctrl-OE group [（1.04±0.52） g]，with a statistically significant difference （t=3.71，P=0.032）； The tumor mass of Lv-RNF43-KD group was （1.94±0.29） g，with no statistically significant difference （t=-1.70，P=0.164） compared with that of the Lv-Ctrl-KD group （1.15±0.74） g. The flow cytometry results showed that the fluorescence intensity of CD8+ T cell perforin in the Lv-RNF43-OE group was 9 034 ± 2 628，which was significantly higher than that in the Lv-Ctrl-OE group （3 847 ±1 637），with a statistically significant difference （t=-3.35，P=0.015）； The fluorescence intensity of CD8+ T cell perforin in the Lv-RNF43-KD group was 966±247，which was significantly lower than that in the Lv-Ctrl-KD group （2 226±646），with a statistically significant difference （t=3.16，P=0.034）； The fluorescence intensity of IFN-γ of CD8+ T cell in the Lv-RNF43-OE group was 2 422±429，which was significantly higher than that of 1 688±324 in the Lv-Ctrl-OE group，with a statistically significant difference （t=-2.73，P=0.034）； The fluorescence intensity of IFN-γ of CD8+ T cell in the Lv-RNF43-KD group was 614 （454，863），with a statistically significant difference （Z=-1.96，P=0.050） compared with 1 159 （1 152，2 068） in the Lv-Ctrl-KD group. The results of quantitative real-time PCR showed that the relative expression level of β-catenin mRNA in the Lv-RNF43-OE group was 0.67±0.16，which was significantly lower than that of 1.00±0.11 in the Lv-Ctrl-OE group，with a statistically significant difference （t=2.98，P=0.041）； The relative expression level of PD-L1 mRNA in the Lv-RNF43-OE group was 0.32±0.09，which was significantly lower than that of 1.00±0.09 in the Lv-Ctrl-OE group，with a statistically significant difference （t=9.13，P=0.001）. The results of the dual-luciferase reporter gene assay showed that the PD-L1 promoter luciferase activity in the pCMV6-NC，RNF43，RNF43+β-catenin and β-catenin groups were 1.00±0.00，0.84±0.00，1.49±0.00 and 1.57±0.03 （F=2 218.33，P<0.001）. Further pairwise comparison showed that compared with the pCMV6-NC group，PD-L1 promoter luciferase activity was significantly lower in the RNF43 group （P<0.001） and significantly higher in the RNF43+β-catenin and β-catenin groups （P<0.001； P=0.003）； compared with the RNF43 group，PD-L1 promoter luciferase activity was significantly higher in the RNF43+β-catenin group （P<0.001）. Conclusion RNF43 may reduce the expression of PD-L1 mRNA in melanoma by inhibiting the expression of β-catenin and promote CD8+ T cell-mediated anti-tumor immune reaction.

Key words: Melanoma, Beta catenin, Ring finger protein 43