过表达的人β防御素-3可抑制结肠癌细胞增殖、迁移和细胞周期

doi:10.5246/jcps.2023.04.022

中国药学(英文版) ›› 2023, Vol. 32 ›› Issue (4): 250-259.DOI: 10.5246/jcps.2023.04.022

过表达的人β防御素-3可抑制结肠癌细胞增殖、迁移和细胞周期

赵炎葱¹, 龚慧媛², 李景华²^,^*()

1. 河南大学第一附属医院, 河南开封 475001
2. 河南大学天然药物与免疫工程重点实验室, 河南开封 475004

收稿日期:2022-11-16 修回日期:2022-12-14 接受日期:2023-01-20 出版日期:2023-04-29 发布日期:2023-04-29
通讯作者: 李景华
作者简介:
+ Tel.: +86-13693894529, E-mail: lijinghua@henu.edu.cn
基金资助:
Postdoctoral Research Grant in Henan Province (Grant No. 202003092), Projects of Science and Technology of Henan Province (Grant No. 222102310671), Key Scientifific Research Projects in Henan Colleges and Universities (Grant No. 22A350012), and the Kaifeng Social Development Science and Technology Project (Grant No. 1503010).

Overexpression of hBD3 inhibits cell proliferation, cell cycle, and migration in colon cancer cells

Yancong Zhao¹, Huiyuan Gong², Jinghua Li²^,^*()

1 The First Affiliated Hospital, Henan University, Kaifeng 475001, China
2 Key Laboratory of Natural Medicine and Immuno-Engineering, Henan University, Kaifeng 475004, China

Received:2022-11-16 Revised:2022-12-14 Accepted:2023-01-20 Online:2023-04-29 Published:2023-04-29
Contact: Jinghua Li

摘要/Abstract

摘要：

本文研究探讨了人β防御素-3(hBD3)对结肠癌HCT116细胞的增殖、迁移和细胞周期的影响。将真核表达载体(pcDNA3.1-hBD3)转染于人结肠癌HCT116细胞。采用qPCR和Western blot方法检测转染效率。使用Western blot方法检测细胞中β-catenin, E-cadherin, N-cadherin蛋白的分子表达水平; 采用单通道Hoechst染色检测细胞核数量和增殖能力; 流式细胞仪检测细胞周期; 划痕法和Transwell法检测结肠癌HCT116细胞的迁移能力。结果显示, 转染过真核表达载体(pcDNA3.1-hBD3)的结肠癌HCT116细胞, hBD3的mRNA表达和蛋白表达水平显著高于结肠癌HCT116和转染pCMV-Blank的HCT116细胞; 其增殖能力和迁移侵袭能力均受到不同程度的抑制; 细胞周期的G2/M期受到阻滞; HCT116-hBD3细胞中β-catenin和N-cadherin两种蛋白的表达水平均低于HCT116和HCT116-Blank细胞, 而E-cadherin蛋白表达的水平均高于HCT116和HCT116-Blank细胞。本文发现, hBD3或许是通过调控Wnt/β-catenin信号通路来调控结肠癌细胞HCT116的增殖、迁移和细胞周期。

关键词: 人β防御素-3, 增殖, 迁移, 结肠癌, HCT116细胞, Wnt/β-catenin信号通路, 细胞周期

Abstract:

In the present study, we investigated the effects of human β-defensin-3 (hBD3) on the proliferation, cell cycle, and migration of HCT116 cells. The eukaryotic expression vector pcDNA3.1-hBD3 was transfected into human colon cancer HCT116 cells. The transfection efficiency was detected by qPCR and Western blotting analysis. The expressions of β-catenin, E-cadherin, and N-cadherin at the protein level were assessed by Western blotting analysis. Single-channel Hoechst33342 was used to detect the number of nuclei and calculate the proportion of proliferating cells. The cell cycle was detected by flow cytometry. The migration ability of HCT116 cells was detected by wound-healing and transwell assays. The results showed that the ability of migration and proliferation was inhibited, and the cell cycle G2/M phase was blocked. The expressions of β-catenin and N-cadherin at the protein level in HCT116-hBD3 cells were lower than those in HCT116 and HCT116-Blank cells, while the expression of E-cadherin at the protein level in HCT116-hBD3 cells was higher than that in HCT116 and HCT116-Blank cells. These findings suggested that hBD3 could regulate the migration and proliferation of HCT116 cells by regulating the Wnt/β-catenin signaling pathway.

Key words: Human β defensin-3, Proliferation, Migration, Colon cancer, HCT116 cells, Wnt/β-catenin signaling pathway, Cell cycle

Supporting:

赵炎葱, 龚慧媛, 李景华. 过表达的人β防御素-3可抑制结肠癌细胞增殖、迁移和细胞周期[J]. 中国药学(英文版), 2023, 32(4): 250-259.

Yancong Zhao, Huiyuan Gong, Jinghua Li. Overexpression of hBD3 inhibits cell proliferation, cell cycle, and migration in colon cancer cells[J]. Journal of Chinese Pharmaceutical Sciences, 2023, 32(4): 250-259.

图/表 6

Figure 1. Establishment of a stable hBD3-expressing colon cancer cell line. (A) Expression levels of hBD3 mRNA in cells by qPCR; (B) Expression levels of hBD3 protein in cells by Western blotting analysis; (C) Statistic analysis of hBD3 protein levels in cells. ***P < 0.001, n = 3.

Figure 2. The effect of hBD3 on proliferation in HCT116 colon cancer cells. (A) Cell proliferation by HCS assay; (B) Statistic analysis of proliferation. **P < 0.01, n = 3.

Figure 3. The effect of hBD3 on G2/M phase arrest in HCT116 colon cancer cells. (A) Detection of cell cycle distribution by flow cytometry; (B) Statistical chart of cell cycle changes; (C) Statistical results of G2M changes. **P < 0.01, ***P < 0.001, n = 3.

Figure 4. Effect of hBD3 on migration in HCT116 colon cancer cells by transwell assay. (A) Detection of the cell migration ability; (B) Statistic analysis of the invasion ability. ***P < 0.001, n = 3.

Figure 5. The effect of hBD3 on migration in HCT116 colon cancer cells by a wound-healing assay. (A) Detection of the migratory ability; (B) Statistic analysis of the invasion ability. **P < 0.01, n = 3.

Figure 6. Expression levels of migration signaling-associated proteins. (A) Expression levels of migration signaling-associated proteins by Western blotting analysis; (B) Statistic analysis of protein levels. ***P < 0.001, n = 3.

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过表达的人β防御素-3可抑制结肠癌细胞增殖、迁移和细胞周期

Overexpression of hBD3 inhibits cell proliferation, cell cycle, and migration in colon cancer cells

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