高表达RNF7增强非小细胞肺癌细胞的PD-1耐药：基于活化NF-kB通路促进CXCL1表达和髓源性抑制细胞的募集

doi:10.12122/j.issn.1673-4254.2024.09.10

摘要/Abstract

摘要：

目的探讨RNF7在非小细胞肺癌（NSCLC）中调控髓源性抑制细胞（MDSCs）的作用机制。方法采用TIMER2.0数据库和免疫组织化学染色分析（IHC）分析RNF7在NSCLC中表达水平。Kaplan-Meier曲线分析RNF7对肺癌患者预后的影响。通过TIMER2.0数据库和多重免疫荧光分析评估RNF7的表达与肿瘤免疫细胞浸润水平之间的相关性。通过C57BL/6小鼠建立皮下移植瘤模型，将RNF7过表达对照组（lenti-GFP，n=10）和RNF7过表达组（lenti-RNF7，n=10），RNF7敲低对照组（Ctrl，n=12）和RNF7敲低组（shRNF7，n=12）的CMT-167细胞分别种植到小鼠皮下，对于RNF7敲低对照组和RNF7敲低组的小鼠7 d后给予anti-PD-1治疗或同型IgG治疗，30 d后获取肿瘤组织，观察皮下瘤体积。通过IHC检测肿瘤组织中S100A8+A9、Gr-1的表达水平，流式细胞术检测肿瘤组织中MDSCs的浸润程度。基因集富集分析（GSEA）RNF7在非小细胞肺癌中可能的调控通路。Western blotting和荧光素酶检测RNF7对NF-κB信号通路的影响。ELISA和RT-qPCR观察趋化因子（C-X-C基序）配体1的表达情况。结果 TCGA数据库分析显示RNF7在NSCLC中的表达水平显著上调，生存分析显示，肺癌患者中RNF7的表达水平越高，患者的预后越差（P<0.001），TIMER2.0数据库显示RNF7的表达与MDSCs的浸润程度呈正相关（P<0.001）。GESA分析提示，在NSCLC中，RNF7高表达富集于NF-κB信号通路。Western blotting实验显示，RNF7低表达能够抑制NF-κB信号通路激活。通过ELISA和RT-qPCR结果显示，敲除RNF7可显著降低CXCL1的转录和翻译（P<0.01）。小鼠皮下移植瘤实验显示，与对照组相比，过表达RNF7可增加MDSCs的浸润程度，敲除RNF7减少MDSCs的浸润程度（P<0.001）。并减轻MDSCs对效应细胞的免疫抑制功能，从而增强了抗PD-1的功效。结论 RNF7通过激活NF-κB信号通路，促进CXCL1的表达，从而使MDSCs趋化募集，使肿瘤产生抗PD-1耐药。

关键词: RNF7, 非小细胞肺癌, NF-κB通路, MDSCs, 抗PD-1治疗

Abstract:

Objective To investigate the mechanism of RNF7 for regulating myeloid-derived suppressor cells (MDSCs) in non-small cell lung cancer (NSCLC). Methods TIMER2.0 database and immunohistochemistry were used to analyze RNF7 expression level and its correlation with immune cell infiltration in non-small cell lung cancer. The impact of RNF7 expression levels on prognosis of lung cancer patients was analyzed using Kaplan-Meier survival analysis. CMT-167 cells with RNF7 overexpression or knockdown were inoculated subcutaneously in C57BL/6 mice, and the mice in RNF7 knockdown group were treated with anti-PD-1 or IgG isotype control 7 days after the inoculation. The tumor tissues were harvested after 30 days for tumor volume measurement, detection of S100A8+A9 and Gr-1 expressions with immunohistochemistry, and analysis of MDSC infiltration. Gene set enrichment analysis (GSEA) was performed to identify the potential pathways regulated by RNF7 in NSCLC. Western blotting and luciferase assays were used to assess the impact of RNF7 on the NF‑κB signaling pathway. ELISA and RT-qPCR were used to measure chemokine (C-X-C motif) ligand 1 (CXCL1) expression. Results RNF7 expression was significantly upregulated in NSCLC, and high RNF7 expression levels were associated with poor prognosis of the patients (P<0.001). TIMER2.0 analysis revealed a positive correlation between RNF7 expression and MDSC infiltration (P<0.001). GSEA suggested that RNF7 was enriched in the NF-κB signaling pathway. In NSCLC cells, RNF7 knockdown significantly inhibited NF-κB activation and reduced CXCL1 expression. In the tumor-bearing mice, RNF7 overexpression significantly increased MDSC infiltration in the tumor tissue, and RNF7 knockdown obviously reduced MDSC infiltration and enhanced the efficacy of anti-PD-1 therapy. Conclusion High expression of RNF7 in NSCLC cells promotes CXCL1 expression by activating the NF-κB signaling pathway, thus leading to the chemotactic recruitment of MDSCs, which contributes to tumor resistance to anti-PD-1 therapy.

Key words: RNF7, non-small cell lung cancer, nuclear factor-κB signaling pathway, myeloid-derived suppressor cells, anti-PD-1

钟娜, 王会杰, 赵文英, 孙珍贵, 耿彪. 高表达RNF7增强非小细胞肺癌细胞的PD-1耐药：基于活化NF-kB通路促进CXCL1表达和髓源性抑制细胞的募集[J]. 南方医科大学学报, 2024, 44(9): 1704-1711.

Na ZHONG, Huijie WANG, Wenying ZHAO, Zhengui SUN, Biao GENG. High RNF7 expression enhances PD-1 resistance of non-small cell lung cancer cells by promoting CXCL1 expression and myeloid-derived suppressor cell recruitment via activating NF-κB signaling[J]. Journal of Southern Medical University, 2024, 44(9): 1704-1711.

图/表 4

图1 RNF7促进肺腺癌进展

Fig. 1 RNF7 promotes lung adenocarcinoma progression. A: TIMER2.0 database analysis showing increased RNF7 mRNA expressions in multiple cancer tissues compared with the adjacent tissues. B: Survival analysis showing a close correlation between high RNF7 expression level and poor prognosis of lung cancer patients. C: RNF7 expression in lung adenocarcinoma tissues and adjacent tissues determined by immunohistochemistry. D: Kaplan-Meier survival analysis showing that patients with high RNF7 expression had shorter overall survival than those with low RNF7 expression (n=90). *P<0.05, **P<0.01, ***P<0.001.

图2 RNF7的表达与MDSCs的浸润呈正相关

Fig.2 RNF7 expression is positively correlated with MDSC infiltration in NSCLC. A: Correlation of RNF7 expression with MDSCs infiltration in pan-cancers predicted by TIMER2.0 database. B: Correlation of RNF7 expression with MDSCs infiltration in lung adenocarcinoma and squamous lung cancer. C: Cox regression analyses using data from TCGA indicating that high MDSC infiltration was significantly associated with poorer prognosis of NSCLC patients. D: Immunofluorescence staining of RNF7 and CD33 in lung adenocarcinoma tissue (DAPI, blue; RNF7, violet; CD33, green). E: Immunohistochemical staining of immune cell markers Gr1 and S100A8+A9 in the tissues (scale bar=50 μm). F: Infiltration of MDSCs assessed by flow cytometry in the tumors. **P<0.01.

图3 RNF7通过促进NF-κB信号通路激活诱导MDSCs的趋化募集

Fig.3 RNF7 induces MDSCs recruitment by promoting NF-κB signaling pathway activation. A: GSEA analysis showing that the high expression of RNF7 in NSCLC was significantly enriched in the NF-κB signaling pathway. B: Western blotting of the key effectors in the NF-κB signaling pathway in cells with RNF7 knockdown. C: Immunoprecipitation of p-IκBα and analysis of its ubiquitination. D: Detection of protein expressions of p-65 and H3 by Western blotting. E: ELISA validation of CXCL1 levels in cell culture supernatants and RT-qPCR of CXCL1 mRNA expression in CMT-167 or H1299 cells. F: Luciferase reporter-based NF-κB activity assay. **P<0.01.

图4 RNF7是NSCLC免疫治疗的潜在治疗靶点

Fig. 4 RNF7 is a potential therapeutic target for immunotherapy of non-small cell lung cancer. A: Dissected tumors from each group of the mice. CMT167 Ctrl or RNF7 knockdown cells were implanted in C57BL/6 (n=5). Isotype control (IgG) or anti-PD-1 were given at 200 µg/mouse on days 7 after cell injection. B: Comparison of tumor volume among the groups. C, D: Immunohistochemical staining of immune cell markers (CD8, Gr1 and S100A8+A9) in different groups (scale bar=50 μm). E: Flow cytometry gating strategy of immune cells and the proportion of MDSCs. **P<0.01.

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