中国癌症杂志 ›› 2020, Vol. 30 ›› Issue (12): 977-983.doi: 10.19401/j.cnki.1007-3639.2020.12.002

• 论著 • 上一篇    下一篇

环状RNA hsa_circ_0050900在乳腺癌中的表达及其对细胞生物学行为影响的机制研究

王晓松,陈俊霞   

  1. 重庆医科大学细胞生物学与遗传学教研室,重庆 400016
  • 出版日期:2020-12-30 发布日期:2021-01-07
  • 通信作者: 陈俊霞 E-mail: chjunxia@126.com
  • 基金资助:
    国家自然科学基金(81672536)。

Expression of circular RNA hsa_circ_0050900 in breast cancer and its effect on cell function

WANG Xiaosong, CHEN Junxia#br#   

  1. Department of Cell Biology and Genetics, Chongqing Medical University, Chongqing 400016, China
  • Online:2020-12-30 Published:2021-01-07
  • Contact: CHEN Junxia E-mail: chjunxia@126.com

摘要: 背景与目的:环状RNA(circular RNA,circRNA)作为特殊的非编码RNA,一般在体内低表达,且不易被RNA酶降解,结构与表达稳定。随着测序技术的进步,目前发现多种肿瘤的发生、发展与circRNA有关。但未见乳腺癌的发生、发展与hsa_circ_0050900的异常表达相关的报道。探究乳腺癌组织中hsa_circ_0050900的表达以及其影响乳腺癌细胞生物学行为的机制。方法:选取在重庆医科大学附属第一医院经手术切除的4例女性乳腺癌组织及对应的癌旁组织,采用RNA测序(RNA sequencing,RNA-seq)进行测序分析,并运用实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR)验证乳腺癌中hsa_circ_0050900的表达,其中包括30例乳腺癌组织及其癌旁组织(在重庆医科大学附属第一医院经手术切除),正常人乳腺上皮细胞系即对照组细胞MCF-10A以及两种乳腺癌细胞系MCF-7、SK-BR-3。通过RTFQ-PCR验证乳腺癌细胞中hsa_circ_0050900被敲低后的表达水平;分别运用细胞计数试剂盒(cell counting kit-8,CCK-8)实验和EdU实验、细胞划痕实验、transwell实验验证细胞的增殖、迁移功能;细胞凋亡的检测采用TUNEL一步法;Hoechst 33342实验通过对细胞核染色确定细胞的状态以检测细胞凋亡;细胞周期蛋白D2(cyclin D2,CCND2)和周期蛋白依赖性激酶4(cyclin-dependent kinase 4,CDK4)的表达采用蛋白质印迹法(Western blot)检测。结果:根据测序结果,挑选出在乳腺癌和细胞中明显高表达的circRNA hsa_circ_0050900;构建的si-circ质粒可显著降低hsa_circ_0050900在乳腺癌细胞中的表达,转染si-circ的细胞增殖能力降低,并促进细胞凋亡,且降低细胞周期相关蛋白CCND2、CDK4的表达。结论:circRNA hsa_circ_0050900在乳腺癌组织中的表达显著高于癌旁组织,敲低hsa_circ_0050900对细胞增殖、迁移能力、细胞凋亡及细胞周期有调控作用。

关键词: 环状RNA, 乳腺癌, hsa_circ_0050900, 增殖, 迁移, 凋亡, 细胞周期

Abstract: Background and purpose: Circular RNA (circRNA), a special non-coding RNA generally low-expressed in vivo, is not easy to be degraded by RNA enzyme, and its structure and expression are stable. Nowadays, with the progress of RNA sequencing, the occurrence and development of a variety of tumors are related to the expression of circRNA. However, there is no report on the relationship between the occurrence and development of breast cancer and the abnormal expression of hsa_circ_0050900. This study aimed to explore the expression of hsa_circ_0050900 in breast cancer and the effect of its abnormal expression on the function of breast cancer cells. Methods: Four cases of female breast cancer and corresponding para-cancerous tissues after clinical operation from the First Affiliated Hospital of Chongqing Medical University were selected and sequenced by RNA sequencing (RNA-seq). Real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) was used to verify the expression of hsa_circ_0050900 in breast cancer and breast cancer cells, including 30 samples of breast cancer tissues and their adjacent tissues (also from the First Affiliated Hospital of Chongqing Medical University), normal human breast epithelial cell line MCF-10A used as control cells, and two breast cancer cell lines MCF-7 and SK-BR-3. The expression level of hsa_circ_0050900 in breast cancer cells was detected by RTFQ-PCR. The proliferation and migration function of breast cancer cells were examined by cell counting kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, wound healing assay and transwell assay. Apoptosis was detected by TUNEL one-step method. Hoechst 33342 test was used to determine the state of cells by nuclear staining for the detection of apoptosis. The expression levels of cyclin D2 (CCND2) and cyclin-dependent kinase 4 (CDK4) were detected by Western blot. Results: Based on the sequencing results, the circRNA hsa_circ_0050900 with high expression in breast cancer tissues and cells was selected. The expression of hsa_circ_0050900 in breast cancer cells was decreased by the constructed si-circ plasmid significantly. The proliferation ability of the cells transfected with si-circ was decreased, apoptosis was promoted, and the expression levels of cell cycle related proteins CCND2 and CDK4 were decreased. Conclusion: The expression of circRNA hsa_circ_0050900 is significantly higher in breast cancer than in para-cancerous tissues. Knocking down hsa_circ_0050900 can regulate cell proliferation, migration, apoptosis and cell cycle.

Key words: CircRNA, Breast cancer, hsa_circ_0050900, Proliferation, Migration, Apoptosis, Cell cycle