CDHR2过表达通过抑制PI3K/Akt通路抑制乳腺癌细胞增殖

doi:10.12122/j.issn.1673-4254.2024.06.12

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (6): 1117-1125.doi: 10.12122/j.issn.1673-4254.2024.06.12

CDHR2过表达通过抑制PI3K/Akt通路抑制乳腺癌细胞增殖

房锦存¹^,²^,³(), 刘立威², 林俊豪¹(), 陈逢生¹^,³()

^1.南方医科大学中西医结合医院肝病科，广东广州 510315
^2.南方医科大学附属何贤纪念医院肿瘤科，广东广州 511400
^3.南方医科大学第三临床医学院，广东广州 510630

收稿日期:2023-11-09 出版日期:2024-06-20 发布日期:2024-07-01
通讯作者: 林俊豪,陈逢生 E-mail:fangjincun@126.com;l_i_n@163.com;fsc0126@163.com
作者简介:房锦存，硕士，主治医师，E-mail: fangjincun@126.com
基金资助:
国家自然科学基金(81872251);广东省自然科学基金(2020A1515010093);南方医科大学中西医结合医院院长基金(1202102002)

Overexpression of CDHR2 inhibits proliferation of breast cancer cells by inhibiting the PI3K/Akt pathway

Jincun FANG¹^,²^,³(), Liwei LIU², Junhao LIN¹(), Fengsheng CHEN¹^,³()

^1.Hepatic Department, Southern Medical University Hospital of Integrated Traditional Chinese and Western Medicine, Southern Medical University, Guangzhou 510315, China
^2.Department of Oncology, Hexian Memorial Affiliated Hospital of Southern Medical University, Guangzhou 511400, China
^3.Third School of Clinical Medicine, Southern Medical University, Guangzhou 510630, China

Received:2023-11-09 Online:2024-06-20 Published:2024-07-01
Contact: Junhao LIN, Fengsheng CHEN E-mail:fangjincun@126.com;l_i_n@163.com;fsc0126@163.com
Supported by:
National Natural Science Foundation of China(81872251)

摘要/Abstract

摘要：

目的研究CDHR2通过PI3K/Akt信号通路抑制乳腺癌细胞生长和细胞周期的作用机制。方法利用生物信息学分析CDHR2在乳腺癌中的表达及生存预后情况，并利用免疫组化验证，采用qRT-PCR、Western blot检测5株乳腺癌细胞株与正常乳腺上皮细胞中CDHR2的表达，并分析CDHR2的表达情况。筛选出CDHR2表达量低的乳腺癌细胞系MDA-MB-231及MCF7进行质粒转染过表达CDHR2，分为NC组（空白质粒对照）和CDHR2组（CDHR2质粒过表达组）。利用CCK-8增殖实验、EdU增殖实验及细胞周期实验探究CDHR2对乳腺癌细胞生长和细胞周期的影响，通过Western blotting检测CDHR2过表达对PI3K/Akt信号通路和周期通路蛋白表达的影响。结果生物信息学分析显示在乳腺癌及癌旁中CDHR2表达量均较低且两者间差异无统计学意义（P>0.05），但高表达CDHR2乳腺癌患者预后更好（P<0.05）。免疫组化、qRT-PCR及Western blot实验提示，CDHR2在乳腺癌组织及乳腺癌细胞中表达均显著下调（P<0.01），CDHR2可以抑制乳腺癌细胞增殖及阻滞乳腺癌细胞的细胞周期（P<0.01），CDHR2能够抑制PI3K和Akt磷酸化蛋白表达、抑制周期蛋白Cyclin D1的表达。结论过表达CDHR2可能通过PI3K/Akt信号通路抑制乳腺细胞生长及阻滞乳腺癌细胞的细胞周期。

关键词: 乳腺癌, CDHR2, PI3K/Akt通路, 增殖, 细胞周期

Abstract:

Objective To investigate the mechanism by which CDHR2 overexpression inhibits breast cancer cell growth and cell cycle pragression via the PI3K/Akt signaling pathway. Methods Bioinformatic analysis was performed to investigate CDHR2 expression in breast cancer and its correlation with survival outcomes of the patients. Immunohistochemistry was used to examine CDHR2 expressions in surgical specimens of tumor and adjacent tissues from 10 patients with breast cancer. CDHR2 expression levels were also detected in 5 breast cancer cell lines and a normal human mammary epithelial cell line using qRT-PCR and Western blotting. Breast cancer cell lines MDA-MB-231 and MCF7 with low CDHR2 expression were transfected with a CDHR2-overexpressing plasmid, and the changes in cell proliferation and cell cycle were evaluated using CCK-8 assay, EdU assay, and cell cycle assay; the changes in expressions of PI3K/Akt signaling pathway and cell cycle pathway proteins were detected with Western blotting. Results Bioinformatic analysis showed low CDHR2 expression level in both breast cancer and adjacent tissues without significant difference between them (P>0.05), but breast cancer patients with a high expression of CDHR2 had a more favorable prognosis. Immunohistochemistry, qRT-PCR and Western blotting showed that the expression of CDHR2 was significantly down-regulated in breast cancer tissues and breast cancer cells (P<0.01), and its overexpression strongly inhibited cell proliferation, caused cell cycle arrest, and significantly inhibited PI3K and Akt phosphorylation and the expression of cyclin D1. Conclusion Overexpression of CDHR2 inhibits proliferation and causes cell cycle arrest in breast cancer cells possibly by inhibiting the PI3K/Akt signaling pathway.

Key words: breast cancer, CDHR2, PI3K/Akt pathway, proliferation, cell cycle

房锦存, 刘立威, 林俊豪, 陈逢生. CDHR2过表达通过抑制PI3K/Akt通路抑制乳腺癌细胞增殖[J]. 南方医科大学学报, 2024, 44(6): 1117-1125.

Jincun FANG, Liwei LIU, Junhao LIN, Fengsheng CHEN. Overexpression of CDHR2 inhibits proliferation of breast cancer cells by inhibiting the PI3K/Akt pathway[J]. Journal of Southern Medical University, 2024, 44(6): 1117-1125.

图/表 11

图1 TCGA数据集分析CDHR2在乳腺癌及癌旁中表达水平

Fig.1 Analysis of CDHR2 expression levels in breast cancer and adjacent tissues based on TCGA dataset.

图2 CDHR2在乳腺癌患者中的生存预后

Fig.2 Survival outcomes of breast cancer patients with low and high CDHR2 expressions. A-D: Relationship between CDHR2 expression and prognosis of the patients with triple-negative (A), luminal A (B), luminal B (C), and Her2-overexpressing (D) breast cancer, respectively.

图3 组织中评估CDHR2 的免疫组织化学(IHC)染色强度

Fig.3 Fig. 3 Immunohistochemical staining for CDHR2 in clinical specimens of breast cancer tissues (Original magnification: ×400). A: Strong expression of CDHR2 in breast tissues. B: Weak expression of CDHR2 in breast tissues. C: Strong expression of CDHR2 in breast cancer tissues. D: Weak expression of CDHR2 in breast cancer tissues. ****P<0.0001.

图4 乳腺癌细胞系以及正常人乳腺上皮细胞中CDHR2的mRNA表达水平

Fig.4 CDHR2 mRNA expression levels in breast cancer cell lines and normal human mammary epithelial cells. **P<0.01, ***P<0.001 vs MCF10A.

图5 乳腺癌细胞系以及正常人乳腺上皮细胞中CDHR2的蛋白表达水平及统计学分析

Fig.5 CDHR2 protein expression levels in breast cancer cell lines and normal human mammary epithelial cells detected by Western blotting. ***P<0.001 vs MCF10A.

图6 过表达质粒转染MDA-MB-231和MCF7细胞系

Fig.6 CDHR2 mRNA expression in MDA-MB-231 and MCF7 cells transfected with CDHR2-overexpressing plasmids. ***P<0.001.

图7 乳腺癌细胞系中CCK-8细胞增殖实验

Fig. 7 CCK-8 cell proliferation assay of the transfected breast cancer cell lines. **P<0.01, ***P<0.001.

图8 乳腺癌细胞系中EdU细胞增殖实验

Fig.8 EdU proliferation assay of the transfected breast cancer cell lines. **P<0.01.

图9 乳腺癌细胞系中细胞周期实验

Fig.9 Flow cytometry for cell cycle analysis of the transfected breast cancer cell lines. **P<0.01.

图10 Western blotting检测CDHR2对PI3K/Akt信号通路和Cyclin D1蛋白表达影响及统计学分析

Fig.10 Western blotting of PI3K/Akt signaling pathway proteins and cyclin D1 in the transfected cells. ***P<0.001, ****P<0.0001 vs negetive control (NC).

图11 分析CDHR2可能存在相互结合作用的蛋白

Fig.11 Proteins with possible binding sites for CDHR2.

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CDHR2过表达通过抑制PI3K/Akt通路抑制乳腺癌细胞增殖

Overexpression of CDHR2 inhibits proliferation of breast cancer cells by inhibiting the PI3K/Akt pathway

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