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Expression of circCEMIP in bladder cancer and its regulatory effects on the proliferation, migration and invasion of UMUC-3 cells
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Abstract:
[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.
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