Y Zhang1, Q Tang, X-M Huang, D-Z Liao. 1. Department of Oncology and Hematology, Affiliated Hospital of Traditional Chinese Medicine of Southwest Medical University, Luzhou, Sichuan, China. liaodazhongmd@sina.com.
Abstract
OBJECTIVE: In recent years, circular RNAs (circRNAs) and microRNAs (miRNAs) have been shown to be related to the development of esophageal squamous cell carcinoma (ESCC). However, their functional mechanisms remain to be investigated. Herein, we focus our research on the functions and mechanisms of circCNOT6L and miR-384 in ESCC. MATERIALS AND METHODS: The levels of circCNOT6L, miR-384, and fibronectin 1 (FN1) were determined using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). RNase R was used to investigate circCNOT6L stabilization. Cell proliferation and apoptosis were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively. Western blot assay was employed to analyze the protein levels of FN1, proliferation-related genes, and iron metabolism-related genes. In addition, the interaction between miR-384 and circCNOT6L or FN1 was predicted by starBase3.0 and confirmed by the Dual-Luciferase reporter assay. Mouse xenograft was carried out to measure the effect of circCNOT6L on tumor growth in vivo. RESULTS: CircCNOT6L and FN1 levels were upregulated, and miR-384 level was downregulated in ESCC tissues/cells. CircCNOT6L knockdown attenuated ESCC cell proliferation and iron metabolism disorder, as well as accelerated apoptosis. Notably, circCNOT6L targeted miR-384, and miR-384 targeted FN1. MiR-384 depletion and FN1 upregulation weakened the effects of circCNOT6L knockdown and miR-384 overexpression on ESCC cell progression, respectively. Besides, circCNOT6L knockdown inhibited tumor growth in vivo. CONCLUSIONS: Our results demonstrated that circCNOT6L positively regulated the development of ESCC cells via modulating miR-384/FN1 axis. Our findings provided a theoretical basis for the therapy of ESCC patients.
OBJECTIVE: In recent years, circular RNAs (circRNAs) and microRNAs (miRNAs) have been shown to be related to the development of esophageal squamous cell carcinoma (ESCC). However, their functional mechanisms remain to be investigated. Herein, we focus our research on the functions and mechanisms of circCNOT6L and miR-384 in ESCC. MATERIALS AND METHODS: The levels of circCNOT6L, miR-384, and fibronectin 1 (FN1) were determined using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). RNase R was used to investigate circCNOT6L stabilization. Cell proliferation and apoptosis were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively. Western blot assay was employed to analyze the protein levels of FN1, proliferation-related genes, and iron metabolism-related genes. In addition, the interaction between miR-384 and circCNOT6L or FN1 was predicted by starBase3.0 and confirmed by the Dual-Luciferase reporter assay. Mouse xenograft was carried out to measure the effect of circCNOT6L on tumor growth in vivo. RESULTS:CircCNOT6L and FN1 levels were upregulated, and miR-384 level was downregulated in ESCC tissues/cells. CircCNOT6L knockdown attenuated ESCC cell proliferation and iron metabolism disorder, as well as accelerated apoptosis. Notably, circCNOT6L targeted miR-384, and miR-384 targeted FN1. MiR-384 depletion and FN1 upregulation weakened the effects of circCNOT6L knockdown and miR-384 overexpression on ESCC cell progression, respectively. Besides, circCNOT6L knockdown inhibited tumor growth in vivo. CONCLUSIONS: Our results demonstrated that circCNOT6L positively regulated the development of ESCC cells via modulating miR-384/FN1 axis. Our findings provided a theoretical basis for the therapy of ESCC patients.