Circ-0007766作为miR-1972海绵调控HER2表达促进乳腺癌细胞迁移和侵袭

赵峻秀; 朱艺; 宋筱羽; 柘钞; 肖雨晗; 刘云多; 李林海; 肖斌

doi:10.19401/j.cnki.1007-3639.2024.10.002

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Circ-0007766作为miR-1972海绵调控HER2表达促进乳腺癌细胞迁移和侵袭

论著 | 更新时间：2025-12-31

- Circ-0007766作为miR-1972海绵调控HER2表达促进乳腺癌细胞迁移和侵袭
- Circ-0007766 acts as a miR-1972 sponge to promote breast cancer cell migration and invasion via upregulation of HER2
- 中国癌症杂志 2024年34卷第10期页码：915-930
- 作者机构：
  
  1. 大理大学公共卫生学院，云南大理 671003
  2. 广州医科大学附属清远医院（清远市人民医院）检验医学部，广东清远 511518
  3. 中国人民解放军南部战区总医院检验科，广东广州 510010
  4. 广州医科大学附属番禺中心医院中心实验室，广东广州 511431
  5. 广东省第二中医院（广东省中医药工程技术研究院）检验科，广州中医药大学第五临床医学院，广东省中医药研究开发重点实验室，广东广州 510095
- 作者简介：
  
  [ "第一作者：赵峻秀（ORCID: 0009-0003-0963-3429），硕士。" ]
  李林海（ORCID: 0000-0002-5924-905X），博士，教授；
  肖斌（ORCID:0000-0002-8822-2542），博士，副教授。
- 基金信息：
- DOI：10.19401/j.cnki.1007-3639.2024.10.002
  中图分类号： R737.9
- 收稿：2024-03-18，
  
  修回：2024-09-10，
  
  纸质出版：2024-10-30
- 稿件说明：
移动端阅览
赵峻秀, 朱艺, 宋筱羽, 等. Circ-0007766作为miR-1972海绵调控HER2表达促进乳腺癌细胞迁移和侵袭[J]. 中国癌症杂志, 2024,34(10):915-930.

Junxiu ZHAO, Yi ZHU, Xiaoyu SONG, et al. Circ-0007766 acts as a miR-1972 sponge to promote breast cancer cell migration and invasion via upregulation of HER2[J]. China Oncology, 2024, 34(10): 915-930.
赵峻秀, 朱艺, 宋筱羽, 等. Circ-0007766作为miR-1972海绵调控HER2表达促进乳腺癌细胞迁移和侵袭[J]. 中国癌症杂志, 2024,34(10):915-930. DOI： 10.19401/j.cnki.1007-3639.2024.10.002.

Junxiu ZHAO, Yi ZHU, Xiaoyu SONG, et al. Circ-0007766 acts as a miR-1972 sponge to promote breast cancer cell migration and invasion via upregulation of HER2[J]. China Oncology, 2024, 34(10): 915-930. DOI： 10.19401/j.cnki.1007-3639.2024.10.002.

摘要

背景与目的：

人表皮生长因子受体2（human epidermal growth factor receptor 2，HER2）是乳腺癌最重要的转移驱动因子之一，20% ~ 30%的乳腺癌患者为HER2高表达型。HER2的表达水平可以在多个分子层面受到调控并且决定了乳腺癌细胞的转移潜能，但HER2的表达如何在mRNA水平受到调控仍不清楚。Circ-0007766是源自HER2的编码基因酪氨酸激酶受体2（tyrosine kinase receptor 2，ERBB2）形成的circRNA，circ-0007766能否通过内源竞争RNA（competing endogenous RNAs，ceRNA）机制调控HER2表达仍鲜见报道。本研究旨在探讨Circ-0007766作为miR-1972海绵调控HER2表达对乳腺癌细胞迁移和侵袭的影响。

方法：

本研究使用高通量circRNA芯片筛选出在HER2阳性乳腺癌细胞中表达特异性高表达的circRNA；采用荧光原位杂交实验（fluorescence

in situ

hybridization，FISH）检测circ-0007766的亚细胞定位；采用microRNA检测原位杂交（BaseScope）实验分析circ-0007766在乳腺癌组织中的表达水平及其临床诊断意义；通过体外转染克隆质粒和siRNA构建过表达和敲低circ-0007766的乳腺癌细胞模型；采用transwell实验评估circ-0007766对乳腺癌细胞迁移和侵袭能力的影响，测定MDA-MB-231与SK-BR-3细胞的迁移和侵袭能力，并用transwell实验评估circ-0007766能否通过 miR-1972促进乳腺癌细胞的迁移和侵袭能力。利用双荧光素酶报告基因检测验证circ-0007766能否通过结合miR-1972调控HER2的表达。通过RNA反义纯化（RNA antisense purification，RAP）实验进一步验证circ-0007766和miR-1972是否具有直接相互作用；在 MDA-MB-231细胞中进行RNA免疫沉淀（RNA immunoprecipitation，RIP）检测，并通过实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RTFQ-PCR）测定 HER2 mRNA的相对3'UTR。采用蛋白质印迹法（Western blot）检测蛋白表达情况。

结果：

circ-0007766在HER2阳性乳腺癌细胞中高表达，circ-0007766分布于细胞质和细胞核，并且主要分布于细胞质。Circ-0007766在乳腺癌组织中的表达水平显著高于癌旁组织，在HER2阳性乳腺癌中表达显著升高，且在HER2阳性乳腺癌样本中的表达水平显著高于HER2阴性样本。在HER2阴性乳腺癌细胞中过表达（在HER2阳性乳腺癌细胞中敲低）circ-0007766能够促进（抑制）乳腺癌细胞的迁移和侵袭能力。Circ-0007766与抑制乳腺癌细胞迁移和侵袭的miR-1972直接结合，形成ceRNA调控网络，抑制由miR-1972介导的HER2 mRNA和蛋白表达水平下调。Circ-0007766能够上调由miR-1972负调控HER2介导的乳腺癌细胞迁移和侵袭的抑制作用。CircRNA通过封存miRNA发挥ceRNA的功能，从而在转录和翻译水平上调控基因表达。

最后，我们发现circ-0007766和HER2在乳腺癌细胞和组织样本中的表达呈正相关，而miR-1972和HER2的表达水平呈负相关。Circ-0007766 能特异性靶向miR-1972，从而阻碍miR-1972对HER2表达的调控作用。

结论：

本研究发现circ-0007766通过miR-1972/HER2信号轴促进乳腺癌细胞的迁移和侵袭，为转移性HER2阳性乳腺癌患者提供了一个新的生物标志物和潜在治疗靶点。

Abstract

Background and purpose:

Human epidermal growth factor receptor 2 (HER2) serves as one of the paramount drivers of breast cancer metastasis

with roughly 20%-30% of breast cancer patients exhibiting high expression of HER2. The expression level of HER2 is regulatable at multiple molecular levels and determines the metastatic potential of breast cancer cells; however

the manner in which HER2 expression is modulated at the mRNA level remains ambiguous. Circ-0007766 is a circRNA originated from the coding gene ERBB2 for HER2

and whether circ-0007766 can regulate HER2 expression via the ceRNA mechanism has not been reported. This study aimed to analyze whether circ-0007766 acts as a miR-1972 sponge to promote breast cancer cell migration and invasion via upregulation of HER2 expression.

Methods:

In this study

a high-throughput circRNA chip was employed to screen for circRNAs that exhibited highly specific expression in HER2-positive breast cancer cells. RNA fluorescence

in situ

hybridization (FISH) was utilized to detect the subcellular localization of circ-0007766. The BaseScope experiment was conducted to analyze the expression level of circ-0007766 in breast cancer tissues and its clinical diagnostic significance. Breast cancer cell models with overexpression and knockdown of circ-0007766 were constructed by transfecting cloning plasmids and siRNA

in vitro

. The effect of circ-0007766 on the migration and invasion of breast cancer cells was assessed using transwell migration and invasion experiments

and the migration and invasion abilities of MDA-MB-231 and SK-BR-3 cells were measured. Additionally

it was evaluated whether circ-0007766 could promot

e the migration and invasion of breast cancer cells through miR-1972. A dual luciferase reporter gene assay was used to verify whether circ-0007766 could regulate HER2 expression by binding to miR-1972. The direct interaction between circ-0007766 and miR-1972 was further verified through the RAP experiment. RIP detection was performed in MDA-MB-231 cells

and the relative 3'UTR of HER2 mRNA was measured by real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR). Western blot was used to detect the protein expressions.

Results:

Circ-0007766 was conspicuously highly expressed in HER2-positive breast cancer cells and distributed in both the cytoplasm and nucleus of cells

with the preponderance being in the cytoplasm. The expression level of circ-0007766 was strikingly higher in breast cancer tissues than in para-cancerous tissues. The expression of circ-0007766 was significantly elevated in HER2-positive breast cancer samples compared with HER2-negative samples. The overexpression (knockdown) of circ-0007766 in HER2-negative breast cancer cells (in HER2-positive breast cancer cells) was capable of promoting (inhibiting) the migration and invasion of breast cancer cells. Circ-0007766 directly bound to miR-1972

which inhibited breast cancer cell migration and invasion

thereby forming an endogenous competitive RNA (ceRNA) regulatory network and impeding the downregulation of HER2 mRNA and protein expression mediated by miR-1972. Circ-0007766 could potentiate the inhibitory effect of miR-1972 on HER2-mediated breast cancer cell migration and invasion that was negatively regulated by miR-1972. CircRNAs sequestered miRNAs to function as ceRNAs

thereby regulating gene expression at both the transcriptional and translational levels. Finally

we discovered that the expressions of circ-0007766 and HER2 were positively correlated in breast cancer cell and tissue samples

while the expression levels of miR-1972 and HER2 were negatively correlated. Circ-0007766 could specifically target miR-1972

to hinder its regulatory effect on HER2 expression.

Conclusion:

This study discovers that circ-0007766 facilitates the migration and invasion of breast cancer cells via the miR-1972/HER2 signal axis

offering a novel biomarker and potential therapeutic target for patients with metastatic HER2-positive breast cancer.

关键词

Keywords

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