作为对抗多种恶性肿瘤的关键防线,自然杀伤(NK)细胞存在于肿瘤微环境(TME)中。据报道,RAD18E3泛素蛋白连接酶(RAD18)可促进多种癌症的恶性进展,但其对NK功能的影响尚未被挖掘。这里,本研究旨在挖掘RAD18调节NK细胞对大肠癌(CRC)细胞杀伤作用的机制.E2F转录因子7(E2F7)和RAD18在CRC组织中的表达,它们的相关性,结合位点,并对RAD18富集途径进行生物信息学分析。通过qRT-PCR和蛋白质印迹测定细胞中E2F7和RAD18的表达。双荧光素酶测定和染色质免疫沉淀(ChIP)测定验证了E2F7和RAD18之间的调控关系。CCK-8测定法用于测定细胞活力,集落形成试验检测细胞增殖,乳酸脱氢酶(LDH)测试以测定NK细胞的细胞毒性,ELISA测定粒细胞-巨噬细胞集落刺激因子(GM-CSF)的水平,肿瘤坏死因子-α(TNF-α)和干扰素-γ(IFN-γ),和免疫荧光检测毒性分子穿孔素和颗粒酶B的表达。在CRC组织和细胞中发现RAD18和E2F7的高表达。沉默RAD18可能会阻碍CRC细胞的增殖,培养NK细胞的活力和细胞毒性,增加GM-CSF的分泌,TNF-α,IFN-γ以及穿孔素和颗粒酶B的表达。ChIP和双荧光素酶报告基因测定确定了RAD18启动子区和E2F7之间的结合关系。E2F7可以激活RAD18的转录,沉默RAD18可以逆转E2F7过表达对NK细胞杀伤的抑制作用。这项工作阐明了E2F7/RAD18轴对CRC中NK细胞杀伤的抑制作用。并为靶向免疫微环境中的CRC免疫治疗提供了新的方向。
As a pivotal defensive line against multitudinous malignant tumors, natural killer (NK) cells exist in the tumor microenvironment (TME).
RAD18 E3 Ubiquitin Protein Ligase (
RAD18) has been reported to foster the malignant progression of multiple cancers, but its effect on NK function has not been mined. Here, the study was designed to mine the mechanism by which RAD18 regulates the killing effect of NK cells on colorectal cancer (CRC) cells. Expression of E2F Transcription Factor 7 (E2F7) and RAD18 in CRC tissues, their correlation, binding sites, and
RAD18 enrichment pathway were analyzed by bioinformatics. Expression of E2F7 and
RAD18 in cells was assayed by qRT-PCR and western blot. Dual-luciferase assay and chromatin immunoprecipitation (ChIP) assay verified the regulatory relationship between E2F7 and RAD18. CCK-8 assay was utilized to assay cell viability, colony formation assay to detect cell proliferation, lactate dehydrogenase (LDH) test to assay NK cell cytotoxicity, ELISA to assay levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), and immunofluorescence to detect expression of toxic molecules perforin and granzyme B. High expression of
RAD18 and E2F7 was found in CRC tissues and cells. Silencing RAD18 could hamper the proliferation of CRC cells, foster viability and cytotoxicity of NK cells, and increase the secretion of GM-CSF, TNF-α, IFN-γ as well as the expression of perforin and granzyme B. Additionally, ChIP and dual-luciferase reporter assay ascertained the binding relationship between RAD18 promoter region and E2F7. E2F7 could activate the transcription of
RAD18, and silencing
RAD18 reversed the inhibitory effect of E2F7 overexpression on NK cell killing. This work clarified the inhibitory effect of the E2F7/RAD18 axis on NK cell killing in CRC, and proffered a new direction for immunotherapy of CRC in targeted immune microenvironment.