TRPV5 TRPV6对结肠癌SW480细胞生物学行为的影响

张慧; 刘模荣; 董辉; 徐靖宇; 谢睿; 文国荣; 刘莲花

doi:10.3969/j.issn.1000-8179.2017.12.114

TRPV5 TRPV6对结肠癌SW480细胞生物学行为的影响

doi: 10.3969/j.issn.1000-8179.2017.12.114

张慧^①,
刘模荣^①, ,,
董辉^②,
徐靖宇^①,
谢睿^①,
文国荣^①,
刘莲花^①

①.
遵义医学院附属医院消化内科（贵州省遵义市563003）
②.
美国加利福尼亚大学医学院胃肠科

基金项目:

贵州省国际科技合作计划项目 G (2014)7014

详细信息

作者简介:
张慧专业方向为早期结肠癌临床及基础研究

通讯作者:
刘模荣 zylmr@163.com

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- 被引次数: 0
出版历程
- 收稿日期: 2017-02-03
- 修回日期: 2017-06-24
- 刊出日期: 2017-06-30

Effect of TRPV5 and TRPV6 channels on the biological behaviors of SW480 colon cancer cell line

ZHANG Hui^①,
LIU Morong^{①
, ,},
DONG Hui^②,
Jingyu XU^①,
Rui XIE^①,
Guorong WEN^①,
Lianhua LIU^①

①.
Department of Gastroenterology, Affiliated Hospital, Zunyi Medical College, Zunyi 563003, China
②.
Division of Gastroenterology, Department of Medicine, University of California, San Diego, School of Medicine, San Diego 92103, USA

Funds:

Guizhou International Science and Technology Cooperation Program G (2014)7014

摘要

摘要: 目的观察TRPV5和TRPV6蛋白表达水平对结肠癌SW480细胞内Ca²⁺水平及细胞增殖、凋亡、迁移等生物学行为的影响。方法给予TRPV5、TRPV6通道激动剂1-25（OH）₂D₃及抑制剂CuCl₂作用结肠癌SW480细胞后，采用免疫组织化学法、Western blot及实时荧光定量PCR技术检测TRPV5、TRPV6蛋白及TRPV5、TRPV6 mRNA表达的变化。使用高速离子成像系统观察结肠癌SW480细胞内Ca²⁺水平的变化；通过MTT法、TUNEL法及划痕修复法观察结肠癌SW480细胞增殖凋亡及迁移的变化。结果 1-25（OH）₂D₃明显上调结肠癌SW480细胞TRPV5、TRPV6 mRNA及蛋白表达水平，使结肠癌SW480细胞内Ca²⁺水平增加，促进结肠癌SW480细胞增殖、迁移及抑制其凋亡（P＜0.05）。CuCl₂明显下调结肠癌SW480细胞中TRPV5、TRPV6蛋白及mRNA表达，使结肠癌SW480细胞内Ca²⁺水平降低（P＜0.05），抑制结肠癌SW480细胞的增殖、迁移及促进细胞的凋亡（P＜0.05）。结论结肠癌SW480细胞中TRPV5和TRPV6蛋白表达水平的变化，能通过调控细胞内Ca²⁺水平影响细胞的增殖、迁移和凋亡等生物学行为。
- 结肠癌 /
- 钙离子通道 /
- TRPV5 /
- TRPV6 /
- Ca²⁺
Abstract: Objective To investigate the role of TRPV5 and TRPV6 in intracellular calcium regulation and biological behaviors of SW480colon cancer cells. Methods qRT-PCR, Western blot, and immunocytochemistry were applied to determine the mRNA and protein expression levels of TRPV5 and TRPV6 in SW480 colon cancer cell line upon treatment with TRPV5 and TRPV6 agonist, 1-25(OH)₂D₃, and inhibitor, CuCl₂. The change of intracellular Ca²⁺ level was examined with a confocal laser scanning microscope. Scratch test, MTT, and TUNEL assays were used to analyze the cell migration, proliferation, and apoptosis, respectively. Results As an agonist of TRPV5 andTRPV6, 1-25(OH)₂D₃ significantly up-regulated the mRNA and protein expression levels of TRPV5 and TRPV6 in SW480 cell lines. On the other hand, CuCl₂, being an inhibitor of TRPV5 and TRPV6, effectively down-regulated the TRPV5 and TRPV6 mRNA and protein expression levels (P < 0.05). The intracellular calcium concentration in SW480 cell line significantly increased upon treatment with 1-25 (OH)2D3, and significantly decreased with CuCl₂ treatment (P < 0.05). 1-25(OH)₂D₃ promoted cell proliferation and migration, and inhibited apoptosis of SW480 cell in a time-and dose-dependent manner (P < 0.05). However, CuCl₂ significantly repressed cell proliferation and migration and induced apoptosis (P < 0.05). Conclusion TRPV5 and TRPV6 can affect the biological behaviors of colon cancerSW480 cells by regulating intracellular Ca²⁺ level.
- colon cancer /
- calcium channel, /
- TRPV5 /
- TRPV6 /
- Ca²⁺

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图 1 免疫组织化学法测定各组结肠癌SW480细胞TRPV5和TRPV6蛋白的表达

Figure 1. Protein expression levels of TRPV5 and TRPV6 in SW480 colon cancer cells by immunocytochemistry

^*: Versus control group, P < 0.05; ^#: versus 1-25(OH)₂D₃ group, P < 0.05

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图 2 Western blot测定各组结肠癌SW480细胞TRPV5和TRPV6蛋白的表达

Figure 2. Protein expression levels of TRPV5 and TRPV6 in SW480 colon cancer cells by Western blot analysis

^*: Versus control group, P＜0.05; ^#: versus 1-25(OH)₂D₃ group, P＜0.05

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图 3 1-25(OH)₂D₃、CuCl₂对结肠癌SW480细胞内Ca²⁺水平的影响（F340 nm×400）

Figure 3. Intracellular Ca²⁺ in SW480 colon cancer cells treated with 1-25(OH)₂D₃ and CuCl₂ (F340nm×400)

A. Control group; B. 1-25(OH)₂D₃ group; C. CuCl₂ group

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图 4 1-25(OH)₂D₃、CuCl₂作用48 h后结肠癌SW480细胞形态（×200）

Figure 4. Effect of 48 h treatment of 1-25(OH)₂D₃ and CuCl₂ on SW480 colon cancer cell morphology

A. Control group; B. 1-25(OH)₂D₃ group; C. CuCl₂ group

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图 5 结肠癌SW480细胞划痕实验

Figure 5. Migration of SW480 colon cancer cell during scratch test

^*: Versus control group, P＜0.05; ^#: versus 1-25(OH)₂D₃ group, P＜0.05

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图 6 结肠癌SW480细胞划痕实验（标尺：500 μm）

Figure 6. Migration of SW480 colon cancer cell scratch test (bar:500 μm)

A. Control group; B. 1-25(OH)₂D₃ group; C. CuCl₂ group

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表 1 TRPV5、TRPV6mRNA在结肠癌SW480细胞中的表达（x±s）

Table 1. Expression levels of TRPV5 and TRPV6 mRNA in SW480 colon cancer cell in terms of mean ± SD

表 2 1-25(OH)₂D₃对结肠癌SW480细胞生长增殖的影响（OD490/增殖率）（n≥3，x±s）

Table 2. Effect of 1-25(OH)₂D₃ on the proliferation of SW480 colon cancer cells (OD490/ proliferation rate, n > 3) in terms of mean ± SD

表 3 CuCl₂对结肠癌SW480细胞生长增殖的影响（OD490/抑制率）（n≥33，x±s）

Table 3. Effect of CuCl₂ on the proliferation of SW480 colon cancer cells (OD490/ inhibiting rate, n > 3) in terms of mean ± SD

表 4 1-25(OH)₂D₃、CuCl₂作用结肠癌SW480细胞48 h后细胞凋亡率的比较（x±s）

Table 4. Comparison of SW480 colon cancer cell apoptosis induced by 1-25(OH)₂D₃ and CuCl₂ for 48 h in terms of mean ± SD

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