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