Identification of miR-199-5p and miR-199-3p Target Genes: Paxillin Facilities Cancer Cell Aggressiveness in Head and Neck Squamous Cell Carcinoma

Abstract

Our previous study revealed that the miR-199 family (miR-199a-5p/-3p and miR-199b-5p/-3p) acts as tumor-suppressive miRNAs in head and neck squamous cell carcinoma (HNSCC). Furthermore, recent studies have indicated that the passenger strands of miRNAs are involved in cancer pathogenesis. The aim of this study was to identify cancer-promoting genes commonly regulated by miR-199-5p and miR-199-3p in HNSCC cells. Our in silico analysis and luciferase reporter assay identified paxillin (PXN) as a direct target of both miR-199-5p and miR-199-3p in HNSCC cells. Analysis of the cancer genome atlas (TCGA) database showed that expression of PXN significantly predicted a worse prognosis (5-year overall survival rate; p = 0.0283). PXN expression was identified as an independent factor predicting patient survival according to multivariate Cox regression analyses (p = 0.0452). Overexpression of PXN was detected in HNSCC clinical specimens by immunostaining. Functional assays in HNSCC cells showed that knockdown of PXN expression attenuated cancer cell migration and invasion, suggesting that aberrant expression of PXN contributed to HNSCC cell aggressiveness. Our miRNA-based approach will provide new insights into the molecular pathogenesis of HNSCC.

Identification of Tumor-Suppressive miR-30e-3p Targets: Involvement of SERPINE1 in the Molecular Pathogenesis of Head and Neck Squamous Cell Carcinoma

“…The miRNA precursors, negative control miRNA, and siRNAs were obtained from Applied Biosystems (Waltham, MA, USA). The procedures used for the transient transfection of miRNAs, siRNAs, and plasmid vectors were described in our previous studies [ 20 , 21 , 57 , 58 ]. miRNAs at 10 nM and siRNAs at 5 nM were transfected into HNSCC cell lines using RNAiMAX reagent (Invitrogen, Waltham, MA, USA).…”

“…The XTT assay (Sigma–Aldrich, St. Louis, MO, USA) characterized cell proliferation, a chamber assay using the Corning BioCoat TM cell culture chamber (Corning, Corning, NY, USA) assessed migration, and Matrigel chamber assays using the Corning BioCoat Matrigel assessed invasion. They were performed with HNSCC cells as described previously [ 20 , 21 , 57 , 58 ]. The reagents used in this study are listed in Supplemental Table S1 .…”

“…RNA extraction from cell lines and qRT-PCR were performed as described in our previous studies [ 20 , 21 , 57 , 58 ]. The TaqMan assays used in this study are summarized in Supplemental Table S1 .…”

“…Western blotting and immunohistochemical procedures were described in our previous studies [ 20 , 21 , 48 , 49 ]. Paraffin sections for immunohistochemistry were obtained from HNSCC patients who underwent surgical treatment at Chiba University hospital.…”

“…However, recent studies showed that some passenger strands can act as oncogenes or tumor suppressors in a wide range of cancers, targeting several cancer-related genes [ 16 , 17 , 18 ]. Moreover, recent studies indicated that both strands of pre-miRNAs cooperated to control oncogenic pathways and exerted tumor-suppressive functions in several cancers [ 19 , 20 , 21 ]. The realization that passenger strands are involved in the molecular pathogenesis of cancer should permit new developments in cancer research.…”