摘要
目的探讨环状RNA(circ_0000437)对乳腺癌细胞功能的影响及其分子机制。方法体外培养人乳腺细胞(MCF-10A)和人乳腺癌细胞(MCF-7和MDA-MB-231),乳腺癌细胞经过分别转染后设置sh-circ_0000437组、mimics组、inhibitor组、si-CTPS1组及其阴性对照组、sh-NC+inhibitor-NC组、sh-circ_0000437+inhibitor-NC组、sh-circ_0000437+inhibitor组、sh-NC+pcDNA-NC组、sh-circ_0000437+pcDNA-NC组、sh-circ_0000437+pcDNA-CTPS1组。qRT-PCR检测乳腺癌细胞株或组织中circ_0000437、let-7b-5p、CTPS1、Notch1、Hes1和Numb的基因表达水平,RNase R检测鉴定circ_0000437的环状结构,亚细胞定位检测显示circ_0000437在乳腺癌细胞中的分布,CCK-8检测细胞增殖能力,Transwell实验检测细胞侵袭能力,划痕实验检测细胞迁移能力,双荧光素酶报告基因和RNA免疫共沉淀实验探究circ_0000437/let-7b-5p/CTPS1间的结合位点,Western blotting检测细胞中CTPS1,E-cadherin、N-cadherin和Vimentin的蛋白表达,小鼠体内成瘤实验验证circ_0000437与CTPS1在体内促癌的作用机制。结果Circ_0000437和CTPS1在乳腺癌组织和细胞系中的表达均上调,let-7b-5p在乳腺癌组织和细胞系中的表达下调(P<0.01),敲低circ_0000437或CTPS1均可抑制乳腺癌细胞的增殖、侵袭、迁移和上皮间质转化(P<0.05),过表达let-7b-5p同样抑制乳腺癌的恶性生物学行为,而抑制let-7b-5p则相反(P<0.05),sh-circ_0000437+pcDNA-NC组的乳腺癌细胞在小鼠体内的生长速度低于sh-NC+pcDNA-NC组,sh-circ_0000437+pcDNA-CTPS1组生长速度高于sh-circ_0000437+pcDNA-NC组(P<0.01),circ_0000437和let-7b-5p、let-7b-5p和CTPS1之间存在结合位点(P<0.01),circ_0000437,let-7b-5p和CTPS1在乳腺癌细胞中存在相互作用(P<0.05)。结论Circ_0000437在乳腺癌组织和细胞中表达上调,且circ_0000437可以通过let-7b-5p/CTPS1轴促进乳腺癌细胞的增殖,侵袭,迁移以及上皮间质转化。
Objective To investigate the role of circular RNA circ_0000437 in regulating biological behaviors of breast cancer cells and the molecular mechanism.Methods Breast cancer MCF-7 and MDA-MB-231 cells were transfected with sh-circ_0000437,mimics,inhibitor,si-CTPS1,or their respective negative controls.qRT-PCR was used to detect the expression levels of circ_0000437,let-7b-5p,CTPS1,Notch1,Hes1,and Numb in breast cancer cell lines and tissues.RNase R digestion was used to confirm the circular structure of circ_0000437 and its subcellular localization in the breast cancer cells was determined by cellular distribution analysis.The changes in proliferation,invasion and migration of the transfected cells were assessed using CCK-8 assay,Transwell assay and scratch assay.Dual-luciferase reporter gene and RNA immunoprecipitation assays were employed to validate binding interactions among circ_0000437,let-7b-5p,and CTPS1.The cellular expressions of CTPS1,Ecadherin,N-cadherin,and vimentin proteins were detected with Western blotting.A tumor-bearing mouse model was used to verify the oncogenic mechanism of circ_0000437 and CTPS1.Results Circ_0000437 and CTPS1 were upregulated while let-7b-5p was downregulated in breast cancer tissues and cell lines.Circ_0000437 or CTPS1 knockdown obviously suppressed breast cancer cell proliferation,invasion,migration and epithelial-mesenchymal transition(EMT).Overexpression of let-7b-5p produced similar inhibitory effects,whereas inhibition of let-7b-5p significantly enhanced malignant behaviors of the cells.In the tumor-bearing mouse models,circ_0000437 knockdown significantly suppressed tumor growth,but co-transfection of the cells with pcDNA-CTPS1 accelerated tumor growth.Binding sites were identified between circ_0000437 and let-7b-5p and between let-7b-5p and CTPS1,and circ_0000437,let-7b-5p,and CTPS1 showed functional interactions in breast cancer cells.Conclusion Circ_0000437 is upregulated in breast cancer tissues and cells,and its high expression promotes proliferation,invasion,migration and EMT of breast cancer cells through the let-7b-5p/CTPS1 axis.
作者
马思源
张博超
浦春
MA Siyuan;ZHANG Bochao;PU Chun(Clinical Laboratory,Xuancheng City Central Hospital,Xuancheng 242000,China;College of Laboratory Medicine,Wannan Medical College,Wuhu 241000,China;Clinical Laboratory,Suixi County Hospital,Huaibei 235100,China)
出处
《南方医科大学学报》
北大核心
2025年第8期1682-1696,共15页
Journal of Southern Medical University
基金
国家级大学生创新创业项目(202210368025)
安徽省省级研究生创新创业实践项目(2022cxcysj184)。
