NLR family CARD domain containing 5 promotes hypoxia-induced cancer progress and carboplatin resistance by activating PI3K/AKT via carcinoembryonic antigen related cell adhesion molecule 1 in non-small cell lung cancer

It is well known that non-small cell lung cancer (NSCLC) is a malignant tumor with high incidence in the world. We aimed to clarify a possible target and identify its precise molecular biological mechanism in NSCLC. NLR family CARD domain containing 5 (NLRC5) is widely expressed in tissues and exerts a vital role in anti-tumor immunity. We determined NLRC5 expression by RT-qPCR and western blot assay. The role of NLRC5 in the development of NSCLC was assessed by a loss-of-function assay. CCK-8, Annexin-V-FITC/PI Apoptosis Detection Kit, Transwell, and wound healing assays were used to determine the cell functions. Drug resistance-related proteins were analyzed by western blot assay. Furthermore, the modulation of NLRC5 on carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) expression and subsequent PI3K/AKT signaling was assessed. In this study, a hyper-expression of NLRC5 was found in NSCLC tissues and cell lines. Knockdown of NLRC5 suppressed cell viability, invasion, and migration, and furthermore promoted cell apoptosis in NSCLC cells. Moreover, under normoxia or hypoxia treatment, the upregulation of NLRC5 was related to carboplatin resistance. NLRC5 silencing increased carboplatin-resistant cell chemosensitivity, as evidenced by the increase in the cell inhibition rate and decrease in drug resistance-related protein expression. Mechanistically, NLRC5 knockdown inhibited the expression of CEACAM1 and subsequently blocked the PI3K/AKT signaling pathway. In conclusion, NLRC5 promotes the malignant biological behaviors of NSCLC cells by activating the PI3K/AKT signaling pathway via the regulation of CEACAM1 expression under normoxia and hypoxia.

众所周知，非小细胞肺癌（non-small cell lung cancer, NSCLC）是全球高发的恶性肿瘤。本研究旨在明确非小细胞肺癌的潜在治疗靶点，并阐明其精准的分子生物学机制。NLR家族含CARD结构域5（NLR family CARD domain containing 5, NLRC5）在多种组织中广泛表达，在抗肿瘤免疫中发挥关键作用。本研究通过实时定量聚合酶链反应（RT-qPCR）和蛋白质印迹（western blot）实验检测NLRC5的表达水平；通过功能丧失实验评估NLRC5在非小细胞肺癌发生发展中的作用。采用细胞计数试剂盒-8（CCK-8）、Annexin-V-FITC/PI细胞凋亡检测试剂盒、Transwell实验及划痕愈合实验检测细胞功能；通过蛋白质印迹实验分析耐药相关蛋白的表达情况。此外，本研究还探究了NLRC5对癌胚抗原相关细胞黏附分子1（carcinoembryonic antigen-related cell adhesion molecule 1, CEACAM1）表达的调控作用，以及其对PI3K/AKT信号通路的后续影响。本研究发现，NLRC5在非小细胞肺癌组织及细胞系中呈高表达状态。敲低NLRC5可抑制非小细胞肺癌细胞的增殖、侵袭与迁移，并促进细胞凋亡。进一步研究显示，无论在常氧还是缺氧条件下，NLRC5的上调均与卡铂耐药相关；沉默NLRC5可提高卡铂耐药细胞的化疗敏感性，表现为细胞抑制率升高，且耐药相关蛋白的表达水平降低。机制上，敲低NLRC5可抑制CEACAM1的表达，进而阻断PI3K/AKT信号通路。综上，在常氧及缺氧条件下，NLRC5可通过调控CEACAM1的表达激活PI3K/AKT信号通路，从而促进非小细胞肺癌细胞的恶性生物学行为。