关键词: 结直肠癌, 钾离子通道调节因子1, 细胞凋亡, 核因子-κB信号通路

Abstract:

Background and purpose: Colorectal cancer (CRC) is one of the major malignant tumors threatening human health worldwide, with long-term high incidence and mortality rate. Potassium channel modulatory factor 1 (KCMF1) is a member of the E3 ubiquitin ligase family. It binds to target proteins through the RING domain and participates in the regulation of a variety of biological processes in vivo. However, the function of KCMF1 in CRC remains unclear. This study aimed to investigate the expression level of E3 ubiquitin ligase KCMF1 in colorectal tumor, and to explore the effects of KCMF1 on the proliferation of CRC cells and its underlying molecular mechanism. Methods: The The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases were used to analyze the expression level of KCMF1 in CRC tissues and adjacent tissues and the association between the KCMF1 expression and the prognosis of CRC patients. Furthermore, immunohistochemical staining was performed to detect the protein level of KCMF1 in 90 paired human CRC tissues and adjacent non-tumor tissues. Lentiviral shRNA delivery system was employed to specifically target the KCMF1 gene (shKCMF1) in HCT116 and HCT15 CRC cell lines. The effects of KCMF1 knockdown on cell proliferation, apoptosis and cell cycle distribution were assessed by methyl thiazoyl terazolium (MTT) assay, colony formation assay, Western blot and flow cytometry. Changes in the transcriptional profile in HCT116 cells upon KCMF1 knockdown were identified by RNA sequencing (RNA-Seq), and the affected signaling pathways were evaluated by bioinformatics analysis. Real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR), Western blot, luciferase reporter assay and cell immunofluorescence assay were utilized to validate the alteration of the affected signaling pathway. Results: The TCGA and GTEx databases and IHC results showed that the mRNA and protein expression levels of KCMF1 in CRC tissues were significantly upregulated compared with adjacent tissues (P<0.01). KCMF1 expression level was negatively correlated with the survival time of patients with CRC (P<0.01), and was positively associated with CRC clinical stage (P<0.05). Compared with control cells, KCMF1 knockdown significantly inhibited the proliferation of HCT116 and HCT15 cells (P<0.001), induced cell apoptosis (P<0.001), and led to cell cycle arrest in G₁ phase (P<0.01). RNA-Seq analysis showed that KCMF1 was involved in the regulation of several signaling pathways, including nuclear factor-κB (NF-κB) signaling pathway. KCMF1 knockdown reduced the transcription levels of the target genes of NF-κB signaling pathway, including BCL-XL, XIAP and CIAP (P<0.05), and suppressed the expression of phosphorylated p65 and nuclear translocation of p65 (P<0.01). Meanwhile, the activity of NF-κB reporter was reduced in tumor cells upon KCMF1 knockdown (P<0.01). Conclusion: The expression of KCMF1 is significantly upregulated in human CRC tissues and positively associated with advanced clinical stage and poor prognosis. KCMF1 may promote the proliferation of CRC cells by activating the NF-κB signaling pathway. KCMF1 may be a potential new therapeutic target for CRC.

Key words: Colorectal cancer, Potassium channel modulatory factor 1, Apoptosis, Nuclear factor-κB signaling pathway