Transcription Factor E2F7 Hampers the Killing Effect of NK Cells against Colorectal Cancer Cells via Activating RAD18 Transcription.

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.