敲低EPHA2通过mTOR磷酸化调控膀胱癌细胞自噬和生物学行为

张永琪; 邹震海; 吴梦琦; 刘贝贝; 郭园园; 刘建民

doi:10.16766/j.cnki.issn.1674-4152.003063

敲低EPHA2通过mTOR磷酸化调控膀胱癌细胞自噬和生物学行为

doi: 10.16766/j.cnki.issn.1674-4152.003063

蚌埠医学院第一附属医院泌尿外科，安徽蚌埠 233004

基金项目:

安徽省卫生健康委科研项目重点项目 AHWJ2021-a007

蚌埠医学院研究生科研创新计划项目 Byycx21081

详细信息

通讯作者:
刘建民，E-mail: LIU-john-jm@sina.com

中图分类号: R737.14 R730.43
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出版历程
- 收稿日期: 2022-11-09
- 网络出版日期: 2023-08-28

Knockdown of EPHA2 regulates autophagy and biological behavior of bladder cancer cell through mTOR phosphorylation

Department of Urology, the First Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui 233004, China

摘要

摘要: 目的探究Si-EPHA2通过靶向mTOR自噬通路干扰膀胱癌细胞生物学行为的分子机制。方法通过Western blotting实验和qRT-PCR实验检测EPHA2在膀胱癌组织和癌旁组织以及膀胱癌细胞(T24)和正常尿路上皮细胞(SV-HUC-1)中的表达水平。通过siRNA转染敲低膀胱癌T24细胞中EPHA2的表达，构成一个抑制EPHA2表达的膀胱癌细胞实验模型，将细胞分成si-NC的空白对照组和si-EPHA2实验组。通过CCK-8实验检测膀胱癌细胞增殖的变化，Transwell实验检测膀胱癌细胞迁移和侵袭的变化，流式细胞学实验检测膀胱癌细胞凋亡的变化；通过Western blotting实验检测敲低EPHA2表达后膀胱癌mTOR磷酸化水平及自噬标记物LC3的表达；在敲低EPHA2表达的T24细胞基础上继续敲低TSC1，检测膀胱癌细胞生物学改变。结果 EPHA2在膀胱癌组织和细胞中表现出较高的表达水平(P＜0.05)；敲低EPHA2降低了mTOR磷酸化水平，自噬水平增加(P＜0.05)；敲低EPHA2显著抑制了膀胱癌细胞的增殖、迁移和侵袭能力(P＜0.05)，促进细胞凋亡(P＜0.05)；在敲低EPHA2表达的基础上，敲低TSC1促进mTOR磷酸化可部分逆转Si-EPHA2对膀胱癌细胞生物学行为的影响(P＜0.05)。结论敲低EPHA2可靶向抑制膀胱癌细胞mTOR磷酸化，增强自噬，抑制膀胱癌细胞的增殖、迁移和侵袭能力，促进膀胱癌细胞凋亡。
- Ephrin A型受体2 /
- 膀胱癌 /
- 雷帕霉素靶蛋白 /
- 自噬
Abstract: Objective To explore the molecular mechanism by which Si-EPHA2 interferes with the biological behaviour of bladder cancer cells by targeting mTOR autophagy pathways. Methods The expression levels of EPHA2 in bladder cancer tissues and paracancerous tissues, as well as bladder cancer cells (T24) and normal urinary tract epithelial cells (SV-HUC-1) were examined by Western blotting assay and qRT-PCR assay. The expression of EPHA2 was knocked down in bladder cancer T24 cells by siRNA transfection to constitute an experimental model of bladder cancer cells with inhibited EPHA2 expression, and the cells were divided into si-NC blank for control group, and si-EPHA2 for experimental group. Changes in bladder cancer cell proliferation were detected by CCK 8 assay, changes in bladder cancer cell migration and invasion by Transwell assay, and changes in bladder cancer cell apoptosis by flow cytometry assay; the level of bladder cancer mTOR phosphorylation level and the expression of autophagy marker LC3 were detected by Western blotting assay after knockdown of EPHA2 expression; Knockdown of TSC1 was continued on top of knockdown of EPHA2-expressing T24 cells to detect changes in bladder cancer cell biology. Results EPHA2 showed higher expression levels in bladder cancer tissues and cells (P < 0.05); knockdown of EPHA2 decreased mTOR phosphorylation levels and increased autophagy levels (P < 0.05); knockdown of EPHA2 significantly inhibited the proliferation, migration and invasion ability of bladder cancer cells (P < 0.05) and promoted apoptosis (P < 0.05); on the basis of knockdown of EPHA2 expression, knockdown of TSC1 to promote mTOR phosphorylation partially reversed the effect of Si-EPHA2 on the biological behavior of bladder cancer cells (P < 0.05). Conclusion Knockdown of EPHA2 can target and inhibit mTOR phosphorylation in bladder cancer cells, enhance autophagy, inhibit the proliferation, migration and invasion ability of bladder cancer cell.
- Ephrin type A receptor 2 /
- Bladder cancer /
- Mammalian target of rapamycin /
- Autophagy

HTML全文

图 1 EPHA2对膀胱癌细胞增殖、迁移、侵袭及凋亡的影响

注：^aP < 0.05。

Figure 1. Effects of EPHA2 on proliferation, migration, invasion and apoptosis of bladder cancer cells

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图 2 EPHA2靶向mTOR轴调控膀胱癌细胞增殖、迁移、侵袭及凋亡

Figure 2. EPHA2 targets the mTOR axis to regulate the proliferation, migration, invasion and apoptosis of bladder cancer cells

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表 1 PCR引物序列

Table 1. PCR primer sequences

基因	引物序列(5’-3’)
EPHA2-F	CTGCTCGCCTGGATT
EPHA2-R	ACGGCTGTGAGGTAGTG
GAPDH-F	GGAGCGAGATCCCTCCAAAAT
GAPDH-R	GGCTGTTGTCATACTTCTCATGG

下载: 导出CSV

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