环状RNA hsa_circ_0008591对乳腺癌细胞生物学行为的影响

doi:10.6040/j.issn.1671-7554.0.2022.1273

摘要/Abstract

摘要： 目的探讨环状RNA(circRNA)hsa_circ_0008591在乳腺癌中的表达及其对乳腺癌细胞生物学行为的影响。方法运用高通量芯片数据分析筛选在乳腺癌组织中差异表达的circRNAs,确定hsa_circ_0008591为研究对象,并通过Sanger测序、核糖核酸酶R(RNaseR)处理等对其进行表征。利用实时荧光定量PCR(qRT-PCR)检测hsa_circ_0008591在乳腺癌组织和细胞中的表达;进一步将质粒或小干扰RNA(siRNA)转染至乳腺癌细胞,采用实时无标记细胞分析技术(RTCA)、CCK-8增殖实验、EdU增殖实验和平板克隆形成实验检测hsa_circ_0008591对细胞增殖能力的影响。运用TargetScan等数据库预测互相作用的微小RNA(miRNA)和蛋白,同时进行功能富集分析;并运用Cytoscape软件绘制竞争性内源RNA(ceRNA)调控网络。结果与癌旁组织比较,hsa_circ_0008591在乳腺癌组织中呈低表达(P=0.007 6);与人正常乳腺上皮细胞比较,hsa_circ_0008591在6种乳腺癌细胞株中的表达水平明显下调(P<0.001)。体外功能实验结果表明,过表达hsa_circ_0008591可抑制MDA-MB-231(P_RTCA<0.001,P_EdU=0.000 6,P_克隆=0.001 0)、MCF-7(P_RTCA<0.001,P_EdU=0.001 7,P_克隆<0.001)细胞的增殖能力,而敲减hsa_circ_0008591可促进SK-BR-3(P_cck-8<0.001,P_EdU<0.001,P_克隆=0.003 4)细胞的增殖能力。下游靶点和通路富集分析结果显示,hsa_circ_0008591可能通过ceRNA机制或与RNA结合蛋白(RBP)相互作用抑制乳腺癌的进展。结论 circRNA hsa_circ_0008591可抑制乳腺癌细胞增殖,有望成为乳腺癌治疗的有效干预靶点。

关键词: 环状RNA, Hsa_circ_0008591, 乳腺癌, 增殖, 生物学行为

Abstract: Objective To investigate the expression of circular RNA(circRNA)hsa_circ_0008591 and its effects on the biological behavior of breast cancer(BC)cells. Methods The differentially expressed circRNAs in BC tissues were screened using high-throughput circRNA microarray, and hsa_circ_0008591 was selected as a candidate, which was characterized by Sanger sequencing and ribonuclease R(RNaseR)treatment. Quantitative real-time PCR(qRT-PCR)was performed to detect the expression of hsa_circ_0008591 in BC tissues and cells. Furthermore, BC cells were transfected with plasmid or small interfering RNA(siRNA)and the effects of hsa_circ_0008591 on cell proliferation were detected with real-time cellular analysis(RTCA), cell counting kit-8(CCK-8)assay, 5-ethynyl-2'-deoxyuridine(EdU)assay, and colony formation assay. The interacting microRNAs(miRNAs)and proteins were predicted by databases such as TargetScan, and function enrichment analysis were performed. A regulatory network of ceRNA was constructed with Cytoscape software. Results Hsa_circ_0008591 was significantly downregulated in BC tissues(P=0.007 6). Compared with normal breast epithelial cells, six BC cell lines had a lower expression of hsa_circ_0008591(P<0.001). In vitro functional experiments showed that overexpression of hsa_circ_0008591 inhibited the proliferation of MDA-MB-231(P_RTCA<0.001, P_EdU=0.000 6, P_colony=0.001 0)and MCF-7 cells(P_RTCA<0.001, P_EdU=0.001 7, P_colony<0.001), while the knockdown of hsa_circ_0008591 promoted the proliferation of SK-BR-3 cells(P_RTCA<0.001, P_EdU<0.001, P_colony=0.003 4). The downstream targets, gene ontology(GO)analysis, and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment indicated that hsa_circ_0008591 inhibited the progression of BC through ceRNA mechanism or interaction with RNA-binding protein(RBP). Conclusion Circular RNA hsa_circ_0008591 can inhibit the proliferation of BC cells and is expected to be an effective intervention target for the treatment of BC.

Key words: Circular RNA, Hsa_circ_0008591, Breast cancer, Proliferation, Biological behavior

中图分类号:

R737.9

董相君,李娟,孔雪,李培龙,赵文静,梁怡然,王丽丽,杜鲁涛,王传新. 环状RNA hsa_circ_0008591对乳腺癌细胞生物学行为的影响[J]. 山东大学学报 (医学版), 2023, 61(2): 78-87.

DONG Xiangjun, LI Juan, KONG Xue, LI Peilong, ZHAO Wenjing, LIANG Yiran, WANG Lili, DU Lutao, WANG Chuanxin. Effects of circular RNA hsa_circ_0008591 on tumor biological behavior of breast cancer cells[J]. Journal of Shandong University (Health Sciences), 2023, 61(2): 78-87.

参考文献

[1] Siegel RL, Miller KD, Fuchs HE, et al. Cancer statistics, 2022 [J]. CA Cancer J Clin, 2022, 72(1): 7-33.
[2] Zheng R, Zhang S, Zeng H, et al. Cancer incidence and mortality in China, 2016[J]. J Natl Cancer Cent, 2022, 2(1): 1-9.
[3] 张雪, 董晓平, 管雅喆, 等. 女性乳腺癌流行病学趋势及危险因素研究进展[J]. 肿瘤防治研究, 2021, 48(1): 87-92. ZHANG Xue, DONG Xiaoping, GUAN Yazhe, et al. Research progress on epidemiological trend and risk factors of female breast cancer [J]. Cancer Research on Prevention and Treatment, 2021, 48(1): 87-92.
[4] 瞿蕾, 唐文静, 吴佳皓. 乳腺癌患者发病的影响因素分析[J]. 中国妇幼健康研究, 2017, 28(5): 505-509. QU Lei, TANG Wenjing, WU Jiahao. Influence factors of patients with breast cancer [J]. Chinese Journal of Woman and Child Health, 2017, 28(5): 505-509.
[5] Dinakar YH, Kumar H, Mudavath SL, et al. Role of STAT3 in the initiation, progression, proliferation and metastasis of breast cancer and strategies to deliver JAK and STAT3 inhibitors [J]. Life Sci, 2022, 309: 120996. doi: 10.1016/j.lfs.2022.120996.
[6] Hu L, Su L, Cheng H, et al. Single-cell RNA sequencing reveals the cellular origin and evolution of breast cancer in BRCA1 mutation carriers [J]. Cancer Res, 2021, 81(10): 2600-2611.
[7] Chiang KC, Yeh CN, Chen HY, et al. PTEN insufficiency modulates ER+ breast cancer cell cycle progression and ncreases cell growth in vitro and in vivo [J]. Drug Des Devel Ther, 2015, 9: 4631-4638. doi: 10.2147/DDDT.S86184.
[8] Keren L, Bosse M, Marquez D, et al. A structured tumor-immune microenvironment in triple negative breast cancer revealed by multiplexed ion beam imaging [J]. Cell, 2018, 174(6): 1373-1387.e19. doi: 10.1016/j.cell.2018.08.039.
[9] Saw PE, Xu X, Chen J, et al. Non-coding RNAs: the new central dogma of cancer biology [J]. Sci China Life Sci, 2021, 64(1): 22-50.
[10] Anastasiadou E, Jacob LS, Slack FJ. Non-coding RNA networks in cancer [J]. Nat Rev Cancer, 2018, 18(1): 5-18.
[11] Chen J, Yang J, Fei X, et al. CircRNA ciRS-7: a novel oncogene in multiple cancers [J]. Int J Biol Sci, 2021, 17(1): 379-389.
[12] Wang X, Fang L. Advances in circular RNAs and their roles in breast cancer [J]. J Exp Clin Cancer Res, 2018, 37(1): 206. doi: 10.1186/s13046-018-0870-8.
[13] Kristensen LS, Jakobsen T, Hager H, et al. The emerging roles of circRNAs in cancer and oncology [J]. Nat Rev Clin Oncol, 2022, 19(3): 188-206.
[14] Pan X, Fang Y, Li X, et al. RBPsuite: RNA-protein binding sites prediction suite based on deep learning [J]. BMC Genomics, 2020, 21(1): 884. doi: 10.1186/s12864-020-07291-6.
[15] Dudekula DB, Panda AC, Grammatikakis I, et al. CircInteractome: a web tool for exploring circular RNAs and their interacting proteins and microRNAs [J]. RNA Biol, 2016, 13(1): 34-42.
[16] Kristensen LS, Andersen MS, Stagsted LVW, et al. The biogenesis, biology and characterization of circular RNAs[J]. Nat Rev Genet, 2019, 20(11): 675-691.
[17] Zhang M, Bai X, Zeng X, et al. circRNA-miRNA-mRNA in breast cancer [J]. Clin Chim Acta, 2021, 523: 120-130. doi: 10.1016/j.cca.2021.09.013.
[18] Xu X, Zhang J, Tian Y, et al. CircRNA inhibits DNA damage repair by interacting with host gene [J]. Mol Cancer, 2020, 19(1): 128. doi: 10.1186/s12943-020-01246-x.
[19] Tang L, Jiang B, Zhu H, et al. The Biogenesis and functions of circRNAs and their roles in breast cancer [J]. Front Oncol, 2021, 11: 605988. doi: 10.3389/fonc.2021.605988.
[20] Xu JZ, Shao CC, Wang XJ, et al. circTADA2As suppress breast cancer progression and metastasis via targeting miR-203a-3p/SOCS3 axis [J]. Cell Death Dis, 2019, 10(3): 175. doi: 10.1038/s41419-019-1382-y.
[21] Misir S, Hepokur C, Aliyazicioglu Y, et al. Circular RNAs serve as miRNA sponges in breast cancer [J]. Breast Cancer, 2020, 27(6): 1048-1057.
[22] Li D, Zhang J, Li J. Role of miRNA sponges in hepatocellular carcinoma [J]. Clin Chim Acta, 2020, 500: 10-19. doi: 10.1016/j.cca.2019.09.013.
[23] Li X, Feng Y, Yang B, et al. A novel circular RNA, hsa_circ_0030998 suppresses lung cancer tumorigenesis and Taxol resistance by sponging miR-558 [J]. Mol Oncol, 2021, 15(8): 2235-2248.
[24] Wang F, Wang X, Li J, et al. CircNOL10 suppresses breast cancer progression by sponging miR-767-5p to regulate SOCS2/JAK/STAT signaling [J]. J Biomed Sci, 2021, 28(1): 4. doi: 10.1186/s12929-020-00697-0.
[25] Zhou WY, Cai ZR, Liu J, et al. Circular RNA: metabolism, functions and interactions with proteins [J]. Mol Cancer, 2020, 19(1): 172. doi: 10.1186/s12943-020-01286-3.
[26] Li J, Sun D, Pu W, et al. Circular RNAs in cancer: biogenesis, function, and clinical significance [J]. Trends Cancer, 2020, 6(4): 319-336.
[27] Yang R, Chen H, Xing L, et al. Hypoxia-induced circWSB1 promotes breast cancer progression through destabilizing p53 by interacting with USP10 [J]. Mol Cancer, 2022, 21(1): 88. doi: 10.1186/s12943-022-01567-z.
[28] Kalmykova S, Kalinina M, Denisov S, et al. Conserved long-range base pairings are associated with pre-mRNA processing of human genes [J]. Nat Commun, 2021, 12(1): 2300. doi: 10.1038/s41467-021-22549-7.
[29] Kanellis DC, Espinoza JA, Zisi A, et al. The exon-junction complex helicase eIF4A3 controls cell fate via coordinated regulation of ribosome biogenesis and translational output [J]. Sci Adv, 2021, 7(32): eabf7561. doi: 10.1126/sciadv.abf7561.
[30] Wei Y, Lu C, Zhou P, et al. EIF4A3-induced circular RNA ASAP1 promotes tumorigenesis and temozolomide resistance of glioblastoma via NRAS/MEK1/ERK1-2 signaling [J]. Neuro Oncol, 2021, 23(4): 611-624.
[31] Yang M, Hu H, Wu S, et al. EIF4A3-regulated circ_0087429 can reverse EMT and inhibit the progression of cervical cancer via miR-5003-3p-dependent upregulation of OGN expression [J]. J Exp Clin Cancer Res, 2022, 41(1): 165. doi: 10.1186/s13046-022-02368-4.
[32] Wang X, Chen M, Fang L. hsa_circ_0068631 promotes breast cancer progression through c-Myc by binding to EIF4A3 [J]. Mol Ther Nucleic Acids, 2021, 26: 122-134. doi: 10.1016/j.omtn.2021.07.003.
[33] Lu C, Rong D, Hui B, et al. CircETFA upregulates CCL5 by sponging miR-612 and recruiting EIF4A3 to promote hepatocellular carcinoma [J]. Cell Death Discov, 2021, 7(1): 321. doi: 10.1038/s41420-021-00710-x.

多维度评价

Viewed

Full text

Abstract

Cited

Shared

Discussed