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
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 promote 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
作者
赵峻秀
朱艺
宋筱羽
柘钞
肖雨晗
刘云多
李林海
肖斌
ZHAO Junxiu;ZHU Yi;SONG Xiaoyu;ZHE Chao;XIAO Yuhan;LIU Yunduo;LI Linhai;XIAO Bin(School of Public Health,Dali University,Dali 671003,Yunnan Province,China;Department of Laboratory Medicine,Qingyuan Hospital Affiliated to Guangzhou Medical University(Qingyuan People's Hospital),Qingyuan 511518,Guangdong Province,China;Clinical Laboratory,General Hospital of Southern Theater Command,Guangzhou 510010,Guangdong Province,China;Central Laboratory,Panyu Central Hospital Affiliated to Guangzhou Medical University,Guangzhou 511431,Guangdong Province,China;Department of Laboratory Medicine,Guangdong Provincial Second Hospital of Traditional Chinese Medicine(Guangdong Provincial Engineering Technology Research Institute of Traditional Chinese Medicine),The Fifth Clinical College of Guangzhou University of Chinese Medicine,Guangdong Key Laboratory of Traditional Chinese Medicine Research and Development,Guangzhou 510095,Guangdong Province,China)
出处
《中国癌症杂志》
CAS
CSCD
北大核心
2024年第10期915-930,共16页
China Oncology
