中国癌症杂志 ›› 2021, Vol. 31 ›› Issue (5): 397-407.doi: 10.19401/j.cnki.1007-3639.2021.05.006

• 论著 • 上一篇    下一篇

外泌体中circ_0044366调控肿瘤相关成纤维细胞MMP2的表达促进结直肠癌转移的机制研究

马维东,王彤彤,朱启航,杨海鸥,林 丹   

  1. 天津医科大学肿瘤医院胰腺肿瘤科,天津 300060
  • 出版日期:2021-05-30 发布日期:2021-05-31
  • 通信作者: 马维东 E-mail: maweidong@tjmuch.com

Mechanism of colorectal cancer cell exosome-derived circ_0044366 in regulating release of MMP2 in fibroblasts and promoting metastasis of colorectal cancer

MA Weidong, WANG Tongtong, ZHU Qihang, YANG Haiou, LIN Dan   

  1. Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China
  • Published:2021-05-30 Online:2021-05-31
  • Contact: MA Weidong E-mail: maweidong@tjmuch.com

摘要: 背景与目的:结直肠癌(colorectal cancer,CRC)是全球癌症死亡的主要原因之一,大量研究发现,环状RNA(circular RNA,circRNA)作为miRNA分子海绵参与CRC细胞的增殖、迁移、侵袭和凋亡等病理生理学过程,circRNA/miRNA/靶基因/靶蛋白轴可能是CRC发生的重要原因之一。探讨CRC细胞来源circ_0044366介导的细胞间通讯调控对CRC进展的影响及其作用机制。方法:选取2018年1月—2019年12月于天津医科大学肿瘤医院诊治的46例CRC患者血清样本为观察组,取同期收治的46例非癌症患者血清样本作为对照组;选取同期CRC手术切除(术前均未接受过放疗和化疗)的CRC患者的肿瘤组织标本和配对癌旁标本40对,利用测序筛选出CRC患者及非癌症人群血清差异表达的环状非编码RNA circ_0044366并预测miR-29b的靶基因。免疫组织化学染色法检测肿瘤组织与癌旁组织中基质金属蛋白酶2(matrix metalloproteinase 2,MMP2)的表达水平。采用双荧光素酶报告基因实验验证靶基因。采用蛋白质印迹法(Western blot)分别检测用SW480细胞外泌体(SW480 exosomes,480 exos)或circ_0044366敲低的SW480细胞外泌体(circ_0044366-deleteSW480 exosomes,480 exos circ_0044366 del)共培养及用miR-29b mimics/inhibitor/NC mimics/inhibitor转染的肿瘤相关成纤维细胞(cancer-associated fibroblast,CAF)的MMP2蛋白的表达水平。采用细胞功能回复实验分别检测同时转染过表达circ_0044366质粒与miR-29b mimics、同时转染降表达circ_0044366质粒与miR-29b inhibitor后MMP2蛋白的表达水平,验证circ_0044366与miR-29b之间的相互调控对MMP2表达的调控作用。采用实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR)检测MMP2 mRNA、circ_0044366和miR-29b的表达水平。建立体内转移模型:给BALB/c裸小鼠分别皮下注射SW480细胞悬液、稳定过表达circ_0044366(circ_0044366-overexpression,circ_0044366-OE)的SW480细胞及敲低circ_0044366(circ_0044366-knockdown,circ_0044366-KD)的SW480细胞悬液,与未移植瘤裸小鼠共计4组,验证circ_0044366对肿瘤迁移的影响。结果:与非癌症人群血清外泌体相比,在CRC血清外泌体中circ_0044366的表达水平与组织中MMP2蛋白的表达水平显著相关(P<0.05)。采用生物信息软件预测circ_0044366与miR-29b的作用靶点及MMP2 mRNA的3’非翻译区(3’-untranslated region,3’-UTR)miR-29b结合位点,通过荧光素酶报告基因实验证实靶点;通过转染上调miR-29b后MMP2蛋白表达明显下降,而下调miR-29b后MMP2蛋白表达明显升高;上调circ_0044366后MMP2蛋白表达明显升高,而下调circ_0044366后MMP2蛋白表达明显下降。细胞功能回复实验结果显示,同时上调或下调circ_0044366和miR-29b时MMP2蛋白表达水平变化不显著。体内实验显示,circ_0044366-KD组瘤体中circ_0044366含量及MMP2蛋白的表达水平降低,而circ_0044366-OE组瘤体中circ_0044366含量及MMP2蛋白的表达水平显著升高。结论:CRC外泌体中circ_0044366转运至CAF中,通过吸附miR-29b促进MMP2表达进而促进肿瘤转移。敲低CRC细胞中的circ_0044366有望通过调控miR-29b/MMP2轴来抑制CRC的侵袭和转移。

关键词: circ_0044366, miR-29b, 外泌体, 结直肠癌, 基质金属蛋白酶2, 转移

Abstract: Background and purpose: Colorectal cancer (CRC) is one of the leading causes of cancer death worldwide. A great deal of studies have found that circular RNA (circRNA), as a molecular sponge of miRNA, participates in the pathological and physiological processes such as proliferation, migration, invasion and apoptosis of CRC cells. The circRNA/miRNA/target gene/target protein axis may be one of the important causes of CRC. The purpose of this study was to investigate the effects of CRC cell-derived circ_0044366-mediated regulation of intercellular communication on the progression of CRC and their mechanisms. Methods: Serum samples of 46 cases of CRC treated in Tianjin Medical University Cancer Institute and Hospital from January 2018 to December 2019 were selected as the observation group, and serum samples of 46 cases of non-cancer population treated in the same period were selected as the control group. A total of 40 tumor tissue samples and paired paracancerous samples from CRC patients undergoing simultaneous CRC surgical resection (both without preoperative radiotherapy and chemotherapy) were selected. The cyclic non-coding RNA circ_0044366 differentially expressed in serum of CRC patients and non-cancer people was screened out by sequencing, and the target gene of miR-29b was predicted. The expression levels of matrix metalloproteinase 2 (MMP2) in the tumor tissues and para-carcinoma tissues were detected by immunohistochemistry. Target genes were verified by dual-luciferase reporter gene assay. Western blot was used to detect the expression levels of SW480 exosomes (480 exos) or circ_0044366-delete SW480 exosomes (480 exos circ_0044366del) and cancer-associated fibroblast (CAF) transfected with MMP2-29b mimics/inhibitors/NC mimics/inhibitors. Cell function recovery assay detected the MMP2 protein expression levels after simultaneous transfection of the overexpressed circ_0044366 plasmid and miR-29b mimics, and simultaneous transfection of the downregulated circ_0044366 plasmid and miR-29b inhibitor, respectively, and verified the regulatory relationship of MMP2 expression by mutual regulation between circ_0044366 and miR-29b. The expression levels of MMP2 mRNA, circ_0044366 and miR-29b were detected by real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR). An in vivo metastasis model was established. Nude mice BALB/c-nu were subcutaneously injected with SW480 cell suspension, circ_0044366-overexpressing (circ_0044366-OE) SW480 cell suspension and circ_0044366-knockdown (circ_0044366-KD) SW480 cell suspension, and paired with xenograft-free nude mice for four groups to verify the effect of circ_0044366 on tumor migration. Results: Compared with serum exosomes of non-cancer population, the expression level of circ_0044366 in CRC serum exosomes was significantly correlated with the expression level of MMP2 protein in tissues (P<0.05). The biological information system software predicted the action target between circ_0044366 and miR-29b and the 3’-untranslated region (3’-UTR) miR-29b binding site of MMP2 mRNA, and confirmed the target through luciferase-reporter gene assay. After upregulating miR-29b by transfection, the expression of MMP2 protein wa significantly decreased, however, it was significantly increased after downregulating miR-29b. The MMP2 protein expression was significantly increased after upregulation of circ_0044366, but significantly decreased after downregulation of circ_0044366. Cell functional recovery assay showed no significant change in MMP2 protein expression level when circ_0044366 and miR-29b were both upregulated or downregulated. The in vivo experiments showed that the circ_0044366 content and MMP2 protein expression in the circ_0044366-KD group were decreased, while the circ_0044366 content and MMP2 protein expression in the circ_0044366-OE group were significantly increased. Conclusion: circ_0044366 in CRC exosomes is transported to CAF and promotes the expression of MMP2 and tumor metastasis by adsorbing miR-29b. Knocking down circ_0044366 in CRC cells is expected to inhibit the invasion and metastasis of CRC by regulating the miR-29b/MMP2 axis.

Key words: circ_0044366, miR-29b, Exosome, Colorectal cancer, Matrix metalloproteinase 2, Metastasis