环状RNA hsa_circ_0001573对乳腺癌细胞生物学行为的影响及机制研究

陈红, 陈俊霞

  1. 重庆医科大学分子医学与肿瘤研究中心，重庆 400016
  • 收稿日期:2022-12-08 修回日期:2023-03-02 出版日期:2023-04-30 发布日期:2023-05-15
  • 通信作者: 陈俊霞
  • 作者简介:陈红（ORCID：0009-0002-7716-9587），硕士研究生在读。

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摘要/Abstract

摘要：

背景与目的：环状RNA（circular RNA，circRNA）是一类呈闭合环状结构的非编码RNA，在基因表达调控方面具有重要的潜能。CircRNA与肿瘤的发生、发展密切相关。本研究旨在探讨hsa_circ_0001573对乳腺癌细胞生物学行为的影响及机制。方法：收集重庆医科大学附属第一医院2020年1月—2021年1月经手术切除的4例乳腺癌组织和癌旁组织，通过RNA微阵列芯片分析（microarray analysis）技术进行分析，并采用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RTFQ-PCR）验证40例乳腺癌组织和癌旁组织以及正常人乳腺上皮细胞MCF-10A和乳腺癌细胞（MCF-7和SK-BR-3）中hsa_circ_0001573的相对表达量。采用荧光原位杂交（fluorescence in situ hybridization，FISH）实验进一步检测其在细胞中的定位与表达。转染干扰质粒后，采用细胞计数试剂盒（cell counting kit-8，CCK-8）、EdU实验检测细胞增殖；通过transwell小室实验、伤口愈合实验和克隆形成实验分别检测细胞侵袭和迁移；采用Hoechst33342、TUNEL与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CCND1、CCND2和CDK4的表达。探讨hsa_circ_0001573对裸鼠移植瘤生长的影响。采用RNA pull down实验检测与hsa_circ_0001573相互作用的蛋白，荧光原位杂交联合免疫荧光（fluorescence in situ hybridization and immunofluorescence，FISH-IF）进一步检测hsa_circ_0001573与GNB4的亚细胞共定位，探究潜在的分子机制。结果：hsa_circ_0001573在乳腺癌组织（P<0.05）和乳腺癌细胞（P <0.001）中显著上调，敲低hsa_circ_0001573能抑制乳腺癌细胞的增殖、迁移和侵袭，诱导细胞凋亡，降低CCND1、CCND2和CDK4的相对表达量。体内实验结果表明，敲低hsa_circ_0001573可抑制移植瘤生长。RNA pull down实验显示，hsa_circ_0001573与GNB4蛋白结合，FISH-IF表明hsa_circ_0001573与GNB4共定位于乳腺癌细胞中，且与GNB4相互作用能促进c-myc的表达。结论：环状RNA hsa_circ_0001573在乳腺癌中高表达，敲低hsa_circ_0001573对细胞增殖、侵袭能力及细胞凋亡和细胞周期有调控作用，并在体内抑制移植瘤生长，可通过与GNB4蛋白的相互作用，本研究结果可望为乳腺癌治疗提供新靶点。

关键词: 环状RNA, 乳腺癌, Hsa_circ_0001573, 细胞增殖, 细胞凋亡, 移植瘤, GNB4

Abstract:

Background and purpose: Circular RNA (CircRNA) is a type of non-coding RNA with a closed circular structure, which has important potential in gene expression regulation. CircRNA is closely related to the occurrence and development of tumor. This study aimed to explore the influence of hsa_circ_0001573 on the biological behavior of breast cancer cells and its mechanism. Methods: Breast cancer tissues and paracancerous tissues of 4 patients surgically removed at the First Affiliated Hospital of Chongqing Medical University from January 2020 to January 2021 were collected, sequenced and analyzed by RNA microarray analysis. Real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) was used to detect the relative expression of hsa_circ_0001573 in breast cancer tissues and paracancerous tissues of 40 cases, as well as normal human breast cancer epithelial cell MCF-10A and breast cancer cells (MCF-7 and SK-BR-3), and the location and expression were further examined by fluorescence in situ hybridization (FISH) assay. The interference vector was transfected into breast cancer cells. Cell counting kit-8 (CCK-8), EdU, wound healing, clonal formation, Hoechst 33342, TUNEL, flow cytometry and transwell assays were adopted to determine cell migration, invasion, proliferation and apoptosis. Western blot was employed to measure the expressions of CCND1, CCND2 and CDK4. The effect of hsa_circ_0001573 on the growth of xenograft tumors was observed in nude mice. RNA pulldown assay was performed to identify hsa_circ_0001573-associated proteins. Subcellular localization of hsa_circ_0001573 and GNB4 was clarified by FISH and immunofluorescence (FISH-IF), to explore potential molecular mechanisms. Results: The expression level of hsa_circ_0001573 was high in breast cancer (P<0.001) and breast cancer cells (P<0.001). Downregulation of hsa_circ_0001573 could inhibit the proliferation, migration and invasion of breast cancer cells, induce cell apoptosis leading to cell cycle arrest in G1 phase, and obviously decrease the expressions of CCND1, CCND2 and CDK4. The results of in vivo experiments showed that knockdown of hsa_circ_0001573 inhibited the growth of xenograft tumors. RNA pulldown experiment showed that hsa_circ_0001573 combined with GNB4 protein. FISH-IF indicated that hsa_circ_0001573 was co-localized with GNB4 in the nucleus and interacted with GNB4 to promote the expression of c-myc. Conclusion: CircRNA hsa_circ_0001573 is highly expressed in breast cancer. Knockdown of hsa_circ_0001573 could regulate cell proliferation, invasion, apoptosis and cell cycle arrest, and inhibit the growth of xenograft tumors in vivo, thus providing a new target for breast cancer treatment.

Key words: CircRNA, Breast cancer, Hsa_circ_0001573, Cell proliferation, Apoptosis, Xenograft tumors, GNB4

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