[摘 要] 目的：探讨环状RNA CEMIP（circCEMIP）对膀胱癌UMUC-3细胞增殖、迁移和侵袭的影响及其分子机制。方法：通 过TCGA数据库分析circCEMIP在膀胱癌组织中的表达水平，分析其表达与膀胱癌患者临床分期及生存期的关系。采用qPCR 法检测circCEMIP在膀胱癌细胞5637、UMUC-3、MGH-U3、J82和T24中的表达。利用RNA干扰技术，分别将si-circCEMIP及其 阴性对照（si-NC）、anti-miR-335及其阴性对照（anti-miR-NC）转染UMUC-3细胞，记为si-circCEMIP组、si-NC组、si-circCEMIP + anti-miR-335 组和 si-circCEMIP + anti-miR-NC 组。采用克隆形成实验、划痕愈合实验和 Transwell 实验分别检测 circCEMIP 和 miR-335表达对UMUC-3细胞增殖、迁移和侵袭能力的影响，双萤光素酶报告基因实验验证circCEMIP与miR-335的靶向关系， WB法检测细胞中VEGF-C信号通路相关蛋白的表达。构建UMUC-3细胞裸鼠皮下移植瘤模型，观察敲低circCEMIP对移植瘤 生长的影响。结果：膀胱癌组织中circCEMIP呈高表达（P < 0.01），其表达水平与膀胱癌的临床分期正相关（P < 0.01）， circCEMIP高表达患者生存率较低（P < 0.01）。circCEMIP在膀胱癌5637、UMUC-3、MGH-U3、J82和T24细胞中呈高表达（均 P < 0.01）。敲低circCEMIP显著降低UMUC-3细胞的增殖、迁移和侵袭能力（均 P < 0.01）。circCEMIP 可靶向结合miR-335 （P < 0.01），敲低circCEMIP能显著上调miR-335表达（P < 0.01）。抑制miR-335表达能逆转敲低circCEMIP对UMUC-3细胞增 殖、迁移和侵袭的抑制作用（均P < 0.01）。敲低circCEMIP能明显下调 VEGF-C 信号通路相关蛋白VEGF-C、MMP-2、MMP-9和 β-catenin表达（均P < 0.01），抑制miR-335表达能部分逆转敲低circCEMIP对该通路相关蛋白表达的抑制作用（均P < 0.01）。体 内实验证实，敲低circCEMIP能够抑制裸鼠膀胱癌移植瘤的生长（P < 0.01）。结论：敲低circCEMIP通过上调miR-335表达抑制 膀胱癌UMUC-3细胞的增殖、迁移和侵袭。

[Abstract] Objective: To study the effects of circular RNA CEMIP (circCEMIP) on the proliferation, migration and invasion of bladder cancer UMUC-3 cells and its molecular mechanism. Methods: Expression of circCEMIP in bladder cancer tissues was analyzed using The Cancer Genome Atlas (TCGA) database, and its correlations with the clinical stage and overall survival of bladder cancer patients were evaluated. The expression levels of circCEMIP in bladder cancer cell lines (5637, UMUC-3, MGH-U3, J82, and T24) were detected by qPCR. Using RNA interference technology, UMUC-3 cells were transfected with si-circCEMIP, negative control (si-NC), anti-miR-335, and negative control (anti-miR-NC), respectively, and assigned into four groups: the si-circCEMIP group, the si-NC group, the si-circCEMIP + anti-miR-335 group, and the si-circCEMIP + anti-miR-NC group. Colony formation assay, wound healing assay, and Transwell assay were used to detect the effects of the expressions of circCEMIP and miR-335 on the proliferation, migration, and invasion of UMUC-3 cells, respectively. Dual-luciferase reporter gene assay was performed to verify the targeting relationship between circCEMIP and miR-335. WB analysis was used to detect the expression of proteins related to the VEGF-C signaling pathway. A UMUC-3 cell nude mouse subcutaneous xenograft model was established to observe the effect of circCEMIP knockdown on the growth of bladder cancer xenografts. Results: circCEMIP was highly expressed in bladder cancer tissues (P < 0.01) and its expression level was positively correlated with the clinical stage of bladder cancer patients (P < 0.01). The survival rate of bladder cancer patients with high expression of circCEMIP was comparatively lower (P < 0.01). circCEMIP in bladder cancer cell lines 5637, UMUC-3, MGH-U3, J82, and T24 was highly expressed (all P < 0.01). circCEMIP knockdown significantly reduced the proliferation, migration, and invasion abilities of UMUC-3 cells (all P < 0.01). circCEMIP can be target bound to miR-335 (P < 0.01), and circCEMIP knockdown could significantly upregulate the expression of miR-335 (P < 0.01). Suppression of miR-335 expression could reverse the inhibitory effect of circCEMIP knockdown on the proliferation, migration, and invasion of UMUC-3 cells (all P < 0.01). Knockdown of circCEMIP could significantly downregulate the expressions of VEGF-C, MMP-2, MMP-9, and β-catenin proteins in the VEGF-C signaling pathway (all P < 0.01). Suppression of miR-335 expression could partially reverse the inhibitory effect of circCEMIP knockdown on the expressions of VEGF-C signaling pathway proteins (all P < 0.01). In vivo experiments confirmed that silencing circCEMIP inhibited the growth of bladder cancer xenografts in nude mice (P < 0.01). Conclusion: Knockdown of circCEMIP suppresses the proliferation, migration and invasion of bladder cancer UMUC-3 cells by upregulating miR-335.

[基金项目] 国家自然科学基金（81960515）