MicroRNA-140-5p Suppresses Tumor Cell Migration And Invasion By Targeting ADAM10-mediated Notch1 Signaling Pathway In Hypopharyngeal Squamous Cell Carcinoma

Background MicroRNAs (miRNAs) are small non-coding RNAs of approximately 22 nucleotides that negatively regulate gene expression at the post-transcriptional level. Recent studies have shown that miRNAs are aberrantly expressed in numerous human cancers, and could function as oncogenes or tumor suppressors. Hypopharyngeal carcinoma is one of the most forms of head and neck squamous cell carcinoma with worst prognosis. Despite the development of various therapies, including surgery, radiotherapy and chemotherapy, the over 5-year survival rate for patients with HSCC is only 30%-35%. The poor prognosis is thought to result from advanced primary disease, local regional recurrence, distant metastasis. Therefore, there is an huge desire to further understand the molecular oncogenic pathways underlying HSCC and to identify related targeted factors of metastasis for treatment. Objective1. To explore the expression of miR-140-5p, ADAM 10 mRNA and Notchl mRNA in hypopharyngeal carcinoma and the clinicopathology significance of miR-140-5p expression in HSCC patients.2. To explore the effect of miR-140-5p on the migration and invasion of FaDu cells.3. Exploration of the molecular mechanism underlying the inhibitory effect of miR-140-5p on the progression of HSCC Methods1. The expressions of miR-140-5p, ADAM 10 mRNA and Notchl mRNA in 40 cases of hypopharyngeal carcinoma tissues and matched normal tissues were detected by real-time quantitative polymerase chain reaction (qRT-PCR).2. Pre-miR-140-5p, anti-miR-140-5p, si-ADAM10 and si-Notchl were transfected by transfection technique. Migration and invasion assays were used to examined the migration and invasion abilities of transfected FaDu cells.3. To experimentally determine whether the 3’UTR of ADAM 10 had an actual target site for miR-140-5p, the luciferase reporter assay was performed.4. FaDu cells were transfected with anti-miR-140-5p and/or si-AD AM10, the western blot assay was performed to test the expression of the Notchl intracellular domain (NICD1).Results1. MiR-140-5p was significantly downregulated in clinical HSCC tissues compared with matched normal tissues. However, the expressions of ADAM 10 mRNA and Notchl mRNA were upregulated in tumor tissues. Remarkably, the downregulation of miR-140-5p was significantly correlated with tumor classification and lymph node metastasis.2. MiR-140-5p inhibits the migration and invasion of FaDu cells in vitro3. Western blot analysis revealed that ADAM 10 protein expression level was markedly reduced in FaDu cells after pre-miR-140-5p transfection. Luciferase activity was significantly inhibited by co-transfection with pre-miR-140-5p and the vector carrying the wide-type 3’UTR of ADAM10.4. Knockdown of ADAM 10 leads to inhibit migration and invasion of FaDu cells.5. NICD1 protein expression significantly increased in FaDu cells transfected with miR-140-5p inhibitor, but decreased in si-ADAM10 transfectants. The co-transfection of miR-140-5p inhibitor with si-AD AM10 significantly abated miR-140-5p inhibitor-induced NICD1 protein expression. Co-transfection with miR-140-5p inhibitor and si-Notchl showed that knockdown of Notchl significantly reversed the increased migration and invasion of FaDu cells induced by miR-140-5p inhibitor.Conclusion:1. MiR-140-5p was significantly downregulated in clinical HSCC tissues. However, the expressions of AD AM 10 mRNA and Notchl mRNA were upregulated in tumor tissues. The downregulation of miR-140-5p was significantly correlated with tumor classification and lymph node metastasis.2. MiR-140-5p inhibited HSCC metastasis by regulating ADAM10-mediated Notchl signaling pathway.3. miR-140-5p may provide new insights into the underlying tumor metastasis as well as a potential therapeutic target in patients with HSCC.