J Cancer 2017; 8(12):2356-2368. doi:10.7150/jca.19470
MicroRNA-204-5p inhibits invasion and metastasis of laryngeal squamous cell carcinoma by suppressing forkhead box C1
1. Department of Otolaryngology, Head & Neck Surgery, The First Hospital, Shanxi Medical University, Taiyuan, Shanxi, China.
2. Shanxi Key Laboratory of Otorhinolaryngology Head and Neck Cancer, Taiyuan, Shanxi, China
#These authors contributed equally to this work.
Background and aim: Understanding the molecular biological mechanisms underlying laryngeal squamous cell carcinoma (LSCC) invasion and metastasis is crucial for diagnosis, treatment, and prognosis. We aimed to examine the expression of the tumor suppressor microRNA-204-5p (miR-204-5p) and its target gene, forkhead box C1 (FOXC1), in human LSCC and explore their roles in the malignant behaviors of LSCC Hep-2 and TU-177 cells.
Methods: The regulatory effects of miR-204-5p on the 3′ untranslated region of FOXC1 predicted by bioinformatics were tested by dual-luciferase reporter assay. Quantitative RT-PCR was used to detect mRNA expression in 43 fresh samples of LSCC and corresponding adjacent normal mucosa (ANM). FOXC1 protein expression was examined by immunohistochemistry. miR-204-5p mimics and FOXC1 siRNA were transfected into LSCC cell lines Hep-2 and TU-177 to observe malignant behavior. miR-204-5p mimics were injected into Hep-2 or TU-177 xenograft tumors in nude mice to examine tumor growth.
Results: The miR-204-5p mRNA level was lower in all 43 LSCC samples than in the ANM samples, but the FOXC1 level was higher in the LSCC samples than in the ANM samples. The miR-204-5p level was lower for stage III and IV cancer and lymph node N+ status samples than for stage I and II cancer and N0 status samples. FOXC1 mRNA and protein levels were higher for N+ than for N0 LSCC. The miR-204-5p mRNA levels were lower in Hep-2 and TU-177 cells than in ANM tissues, but FOXC1 mRNA levels were higher in Hep-2 and TU-177 cells than in ANM tissues. Dual-luciferase reporter assays demonstrated the targeted regulatory effects of miR-204-5p on the FOXC1 3′ UTR. Cell proliferation and colony formation was facilitated with miR-204-5p mimics and FOXC1 siRNA, with weaker cell migration and invasion than the controls. Moreover, miR-204-5p overexpression or FOXC1 knockdown inhibited the EMT process in LSCC cells. In vivo experiments demonstrated that injection of miR-204-5p into Hep-2 and TU-177 xenograft tumors in nude mice significantly inhibited tumor growth.
Conclusions: miR-204-5p is involved in the invasion and metastasis of LSCC. It has a targeted regulatory effect on FOXC1 expression; malignant LSCC behaviors, including cell proliferation, invasion, and metastasis, are suppressed, and tumor growth in vivo is inhibited. This suggests that miR-204-5p may be a target for molecular therapy of LSCC in the future.
Keywords: hsa-miR-204-5p, FOXC1, laryngeal squamous cell carcinoma, invasion, metastasis
Gao W, Wu Y, He X, Zhang C, Zhu M, Chen B, Liu Q, Qu X, Li W, Wen S, Wang B. MicroRNA-204-5p inhibits invasion and metastasis of laryngeal squamous cell carcinoma by suppressing forkhead box C1. J Cancer 2017; 8(12):2356-2368. doi:10.7150/jca.19470. Available from http://www.jcancer.org/v08p2356.htm