环状RNA hsa_circ_0058514在三阴性乳腺癌中的表达及作用研究

背景与目的:环状RNA(circular RNA,circRNA)是一类具有重要调节潜能的非编码RNA,参与多种肿瘤的发生、发展,但对于三阴性乳腺癌(triple-negative breast cancer,TNBC)尚未见报道。该研究旨在探讨环状RNA hsa_circ_0058514在TNBC发生、发展中的作用。方法:采用RNA测序(RNA sequencing,RNA-seq)对4对TNBC组织和癌旁组织进行分析,采用实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)对20对TNBC组织和癌旁组织以及正常人乳腺上皮细胞MCF-10A和TNBC细胞MDA-MB-231和BT-549中hsa_circ_0058514的表达进行验证。将干扰质粒pLL3.7-sh-circ转染TNBC细胞MDA-MB-231和BT-549后,采用RTFQ-PCR检测细胞中hsa_circ_0058514的表达;采用细胞计数试剂盒(cellcountingkit-8,CCK-8)和EdU实验检测细胞增殖;采用划痕和Transwell小室实验分别检测细胞迁移和侵袭;采用流式细胞术检测细胞凋亡和细胞周期;采用蛋白质印迹法(Western blot)检测细胞周期蛋白E1(cyclin E1,CCNE1)和细胞周期蛋白依赖性激酶2(cyclin-dependent kinase 2,CDK2)蛋白的表达。结果:环状RNA hsa_circ_0058514在TNBC组织和细胞中显著高表达(P<0.001,P<0.01);转染pLL3.7-sh-circ后,TNBC细胞中hsa_circ_0058514的表达量显著低于对照组(P<0.001)。下调hsa_circ_0058514后,TNBC细胞增殖、迁移及侵袭能力下降,并促进细胞凋亡,导致细胞周期阻滞。Western blot结果显示,转染pLL3.7-sh-circ后,CCNE1和CDK2蛋白的表达下调(P<0.05)。结论:环状RNA hsa_circ_0058514在TNBC组织和细胞中均高表达,其在TNBC发生、发展中可能起到癌基因的作用,并有望成为TNBC治疗的新靶点。

Effects of circular RNA hsa_circ_0058514 on the development and progression of triple-negative breast cancer

Background and purpose: Circular RNA (circRNA) is a class of non-coding RNAs with important regulatory potential. It is involved in the development and progression of various cancers, but has not been reported in triple-negative breast cancer. The aim of the present study was to investigate the effects of circRNA hsa_circ_0058514 on the development and progression of triple-negative breast cancer. Methods: RNA sequencing (RNA-seq) was performed on 4 pairs of triple-negative breast cancer tissues and adjacent non-cancerous tissues. The relative expression levels of hsa_circ_0058514 in 20 pairs of triplenegative breast cancer tissues and adjacent non-cancerous tissues, normal breast epithelial cell MCF-10A and triple-negative breast cancer MDA-MB-231 and BT-549 cells were detected by real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR). The interference vector pLL3.7-sh-circ was transfected into triple-negative breast cancer MDA-MB-231 and BT-549 cells, and the expression level of hsa_circ_0058514 was determined by RTFQ-PCR. The proliferations of MDA-MB-231 and BT-549 cells were detected by cell counting kit-8 (CCK-8) and EdU assays. The abilities of migration and invasion of MDA-MB-231 and BT-549 cells were examined by wound healing assay and Transwell assay, respectively. The cell apoptosis and cell cycle distribution were analyzed by flow cytometry. Western blot was employed to observe the protein expression levels of CCNE1 and CDK2. Results: The expression levels of circRNA hsa_circ_0058514 in triple-negative breast cancer tissues and cells were significantly up-regulated (P<0.001, P<0.01). The expression of hsa_circ_0058514 in the group transfected with interference vector was much lower than that in the control group. Downregulation of hsa_circ_0058514 decreased the abilities of proliferation, migration and invasion of triple-negative breast cancer cells, leading to cell apoptosis and cell cycle arrest. And lower protein expression levels of CCNE1 and CDK2 were observed by Western blot after transfection of hsa_circ_0058514 (P<0.05). Conclusion: The expression of circRNA hsa_circ_0058514 is significantly upregulated in triple-negative breast cancer tissues and MDA-MB-231 and BT-549 cells. It may serve as an oncogene in the development and progression of triple-negative breast cancer, and it is expected to become a new target for the treatment of triple-negative breast cancer.