Abstract
Hypopharyngeal squamous cell carcinoma (HSCC) accounts 95% of hypopharyngeal cancer, which is characterized by high early metastasis rate and poor prognosis. It is reported that circular RNA is involved in the occurrence and development of cancer; however, the role of circRNA in hypopharyngeal cancer has little been investigated. We performed hypopharyngeal carcinoma circRNA microarray and qRT-PCR verification. The results showed circ_0058106 expression level was significantly upregulated in tumor tissues than in corresponding normal tissues. We found that circ_0058106 upregulation promoted proliferation, migration and invasion of HSCC cells, while knockdown of circ_0058106 inhibited proliferation, migration and invasion of HSCC cells both in vitro and in vivo. Bioinformatics predicted circ_0058106 may interact with miR-185-3p. We verified circ_0058106 directly bound miR-185-3p and downregulated miR-185-3p expression by using dual-luciferase reporter assay and qRT-PCR. Moreover, we proved circ_0058106 promoted HSCC cells tumorigenesis and EMT process by regulating Wnt2b/β-catenin/c-Myc pathway via miR-185-3p. In conclusion, our findings firstly confirmed the carcinogenic effect of circ_0058106 in promoting HSCC cells tumorigenesis, metastasis, invasion and EMT process by regulating Wnt2b/β-catenin/c-Myc pathway through sponging miR-185-3p, indicating that circ_0058106 may be a new therapeutic target and prognostic marker for HSCC.
Highlights
Hypopharyngeal cancer is a malignant tumor derived from the epithelial tissue of the laryngeal mucosa, accounts for about 3% of head and neck malignancies [1]
Our results suggest that circ_0058106 could promote Hypopharyngeal squamous cell carcinoma (HSCC) proliferation, migration, invasion and EMT process by regulating Wnt2b/β-catenin/c-Myc pathway via direct binding to miR-185-3p
CircRNA microarray was performed in the four paired HSCC tumor and adjacent normal tissues
Summary
Cell lines and cell culture Fadu, TU686, TU212 and Detroit562 cells were obtained from American Type Culture Collection and identified by STR profiling. Cells were cultured in DMEM high glucose (BasalMedia, Shanghai, China) containing 10% fetal bovine serum (Sigma, Saint Louis, USA). 105 cells were seeded into the upper Matrigel-coated invasion chamber and cultured in serum-free medium. Digested cells in the logarithmic growth phase and inoculated 300 cells (for Fadu) or 500 cells (for TU212) in each 6-well plate, cultured in DMEM medium with 10% FBS for 2 weeks; aspirated the supernatant and washed 3 times by using PBS; fixed with methanol for 30 min and stained with crystal violet for 30 min. Fadu cells were seeded on a 48-well plate, cultured for 24 h, co-transfected with wildtype or mutant pmiRGlo-circ_0058106 (or pmiRGlo-Wnt2b) dual-luciferase reporter vectors incorporating miR-185-3p binding sites vectors and miR185-3p mimics or negative control by using transfection reagents. A two-tailed Student’s t test was used to evaluate the statistical differences between
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