Mechanism study of KCMF1 promoting proliferation and NF-κB signaling transduction in colorectal cancer cells

Zhibai WU; Guiqin XU; Li ZHANG; Zhaojuan YANG; Yun LIU; Kun JIAO; Zehong CHEN; Chen XU; You ZUO; Ningqian ZHENG; Zhiqian YE; Yongzhong LIU

doi:10.19401/j.cnki.1007-3639.2024.11.001

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Mechanism study of KCMF1 promoting proliferation and NF-κB signaling transduction in colorectal cancer cells

Article | 更新时间：2025-12-31

- Mechanism study of KCMF1 promoting proliferation and NF-κB signaling transduction in colorectal cancer cells
- China Oncology Vol. 34, Issue 11, Pages: 987-997(2024)
- 作者机构：
  
  1. 上海交通大学医学院附属仁济医院，上海市肿瘤研究所，肿瘤系统医学全国重点实验室，上海 200032
  2. 上海交通大学生物医学工程学院，上海市肿瘤研究所，肿瘤系统医学国家重点实验室，上海 200032
- 作者简介：
- 基金信息：
- DOI：10.19401/j.cnki.1007-3639.2024.11.001
  CLC： R735.3
- Received：26 June 2024，
  
  Revised：2024-09-12，
  
  Published：30 November 2024
- 稿件说明：
移动端阅览
Zhibai WU, Guiqin XU, Li ZHANG, et al. Mechanism study of KCMF1 promoting proliferation and NF-κB signaling transduction in colorectal cancer cells[J]. China Oncology, 2024, 34(11): 987-997.
DOI：

Zhibai WU, Guiqin XU, Li ZHANG, et al. Mechanism study of KCMF1 promoting proliferation and NF-κB signaling transduction in colorectal cancer cells[J]. China Oncology, 2024, 34(11): 987-997. DOI： 10.19401/j.cnki.1007-3639.2024.11.001.

摘要

背景与目的：

结直肠癌（colorecta

l cancer，CRC）是危害全球人类生命健康的主要恶性肿瘤之一，其发病率和死亡率长期居高不下。钾离子通道调节因子1（potassium channel modulatory factor 1，KCMF1）属于E3泛素连接酶家族的一员，通过RING结构域与靶蛋白结合，参与调节体内多种生物学过程。然而，KCMF1在CRC中的作用尚不清楚。本研究旨在探索KCMF1在CRC中的表达情况，并探讨其对CRC细胞增殖的影响及可能的分子机制。

方法：

利用癌症基因组图谱（The Cancer Genome Atlas，TCGA）和基因型-组织表达（Genotype-Tissue Expression，GTEx）数据库，分析CRC组织中KCMF1的表达水平及其与CRC患者预后的相关性。通过免疫组织化学法（immunohistochemistry，IHC）检测90例配对的人CRC组织样本中KCMF1的蛋白表达水平。通过慢病毒感染肠癌HCT116和HCT15细胞，转导针对

KCMF

1基因的短发夹RNA（shKCMF1），分别采用四甲基偶氮唑盐（methyl thiazoyl terazolium，MTT）实验和克隆形成实验检测敲降

KCMF

1对细胞增殖的影响；采用蛋白质印迹法（Western blot）和流式细胞学实验检测敲降

KCMF

1对细胞凋亡和细胞周期的影响；利用转录组测序（RNA sequencing，RNA-Seq）检测敲降

KCMF

1对HCT116细胞的转录谱的影响，运用生物信息学分析受KCMF1调控的信号通路；利用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RTFQ-PCR）、Western blot、荧光素酶报告基因实验及细胞免疫荧光实验等验证相关信号通路的改变。

结果：

TCGA和GTEx数据库分析以及IHC结果显示，与癌旁组织相比，CRC组织中KCMF1 mRNA的表达及蛋白水平均显著升高（

0.01），其表达水平与患者的生存时间呈负相关（

0.01），并与CRC临床分期呈正相关（

0.05）。与对照细胞相比，敲降

KCMF

1的HCT116和HCT15细胞的增殖能力显著降低（

0.001），细胞凋亡水平显著增高（

0.001），细胞周期停滞在G

期（

0.01）。RNA-Seq分析发现，KCMF1参与调控核因子-κB（nuclear factor-κB，NF-κB）等多个信号通路。敲降

KCMF

1后，NF-κB信号通路下游靶基因

BCL

、

XIAP

和

CIAP

的转录水平降低（

0.05），p65的磷酸化水平下降，同时p65的核转移受抑制（

0.01），NF-κB信号报告基因活性降低（

0.01）。

结论：

KCMF1在人CRC组织中呈高表达，并与患者的高临床分期和不良预后呈正相关；KCMF1可能通过激活NF-κB信号通路促进CRC细胞增殖。KCMF1可能是CRC的一个潜在治疗新靶点。

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

KCMF

1 gene (shKCMF1) in HCT116 and HCT15 CRC cell lines. The effects of

KCMF

1 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

KCMF

1 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 (

0.01). KCMF1 expression level was negatively correlated with the survival time of patients with CRC (

0.01)

and was positively associated with CRC clinical stage (

0.05). Compared with control cells

KCMF

1 knockdown significantly inhibited the proliferation of HCT116 and HCT15 cells (

0.001)

induced cell apoptosis (

0.001)

and led to cell cycle arrest in G

phase (

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.

KCMF

1 knockdown reduced the transcription levels of the target genes of NF-κB signaling pathway

including

BCL

XIAP

and

CIAP

(

0.05)

and suppressed the expression of phosphorylated p65 and nuclear translocation of p65 (

0.01). Meanwhile

the activity of NF-κB reporter was reduced in tumor cells upon

KCMF

1 knockdown (

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.

关键词

Keywords

references

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