Hsa_circ_0000825 promotes the progression of laryngeal squamous cell carcinoma by sponging miR-766 and interacting with ELAVL1.

ABSTRACT

Emerging evidence suggests that circular RNAs (circRNAs) are involved in the regulation of tumourigenesis and progression of a variety of malignant tumours. In this study, we aimed to identify laryngeal squamous cell carcinoma (LSCC)-specific circRNAs and explore their biological functions and underlying molecular mechanisms. Employing microarray and qRT-PCR, hsa_circ_0000825 was found to be significantly increased in LSCC tissues versus para-cancerous tissues. High hsa_circ_0000825 expression was positively associated with advanced clinical stages, lymph node metastasis, and poor survival. Furthermore, the overexpression of hsa_circ_0000825 in TU177 and AMC-HN-8 cells promoted cell proliferation. Transwell assays showed enhanced migration and invasion of TU177 and AMC-HN-8 cells upon overexpression of hsa_circ_0000825. Conversely, the knockdown of hsa_circ_0000825 had the opposite effect. Xenograft tumours in BALB/c nude mice derived from hsa_circ_0000825-overexpressed TU177 cells showed greater volume and weight than those derived from control TU177 cells. Mechanistically, nuclear-cytoplasmic fractionation assay confirmed that hsa_circ_0000825 was mainly located in the cytoplasm of TU177 and AMC-HN-8 cells. The AGO2-RNA immunoprecipitation (RIP) assay revealed that hsa_circ_0000825 was significantly enriched in the AGO2-precipitated complex in both TU177 and AMC-HN-8 cells, suggesting that this circRNA may function via a competitive endogenous RNA (ceRNA) mechanism. Next, bioinformatics analysis, biotinylated-oligo pull-down assay and dual-luciferase reporter assay verified that miR-766 could be sponged by hsa_circ_0000825 and also target 3'UTR of HOXD10 mRNA. Moreover, miR-766 was shown to be involved in the pro-oncogenic effect of hsa_circ_0000825. This occurred via the mediation of hsa_circ_0000825-enhanced HOXD10 mRNA by the ceRNA mechanism in TU177 and AMC-HN-8 cells. Besides, RNA-binding protein (RBP) ELAVL1 interacted with hsa_circ_0000825 in TU177 and AMC-HN-8 cells, as revealed through bioinformatics analysis, biotinylated-oligo pull-down assays, and RIP assays. ELAVL1 knockdown decreased cell proliferation by 38 % and 34 % in hsa_circ_0000825-overexpressed TU177 and AMC-HN-8 cells (P < 0.05). Similarly, ELAVL1 was involved in the pro-migration and pro-invasion effects of hsa_circ_0000825 overexpression. In addition, comprehensive analysis of mRNA-seq in hsa_circ_0000825-overexpressed TU177 cells, as well as catRAPID and TCGA databases, suggested that ITGB2, HOXD10, and MTCL1 might be crucial downstream target mRNAs of ELAVL1 in LSCC, participating in the hsa_circ_0000825-ELAVL1 axis pro-oncogenic effect. Taken together, hsa_circ_0000825 plays a pro-oncogenic role in LSCC via the miR-766/HOXD10 axis and ELAVL1 and may serve as a promising specific biomarker and therapeutic target for LSCC.