Impact of miR-1/miR-133 Clustered miRNAs: PFN2 Facilitates Malignant Phenotypes in Head and Neck Squamous Cell Carcinoma

Abstract

Based on our original RNA sequence-based microRNA (miRNA) signatures of head and neck squamous cell carcinoma (HNSCC), it was revealed that the expression levels of miR-1-3p, miR-206, miR-133a-3p, and miR-133b were significantly suppressed in cancer specimens. Seed sequences of miR-1-3p/miR-206 and miR-133a-3p/miR-133b are identical. Interestingly, miR-1-3p/miR-133a-3p and miR-206/miR-133b are clustered in the human genome. We hypothesized that the genes coordinately controlled by these miRNAs are closely involved in the malignant transformation of HNSCC. Our in silico analysis identified a total of 28 genes that had putative miR-1-3p/miR-133a-3p and miR-206/miR-133b binding sites. Moreover, their expression levels were upregulated in HNSCC tissues. Multivariate Cox regression analyses showed that expression of PFN2 and PSEN1 were independent prognostic factors for patients with HNSCC (p < 0.05). Notably, four miRNAs (i.e., miR-1-3p, miR-206, miR-133a-3p, and miR-133b) directly bound the 3′untranslated region of PFN2 and controlled expression of the gene in HNSCC cells. Overexpression of PFN2 was confirmed in clinical specimens, and its aberrant expression facilitated cancer cell migration and invasion abilities. Our miRNA-based strategy continues to uncover novel genes closely involved in the oncogenesis of HNSCC.

Identification of Antitumor miR-30e-5p Controlled Genes; Diagnostic and Prognostic Biomarkers for Head and Neck Squamous Cell Carcinoma

“…Overexpression of PFN2 was reported in several types of cancers, including HNSCC [ 42 ]. Recently, we revealed that PFN2 was directly regulated by tumor-suppressive miR-1 / miR-133 clustered miRNAs in HNSCC cells, and its overexpression promoted cancer cell malignant transformation [ 25 ]. Another study showed that overexpression of PFN2 enhanced the aggressiveness of small cell lung cancer (SCLC), including cell proliferation, migration, and invasion.…”

“…The protocols used for transient transfection of miRNAs and siRNAs were described in our previous studies [ 23 , 24 , 25 , 26 ]. The miRNA precursors and siRNAs used in this report were detailed in Table S2 .…”

“…The procedures for conducting functional assays (cell proliferation, migration, and invasion assays) with HNSCC cells were described in earlier publications [ 23 , 24 , 25 , 26 ]. In brief, for proliferation assays, Sa3 or SAS cells were transferred to 96-well plates at 3.0 × 10 3 cells per well.…”

“…The procedures for plasmid construction and the dual-luciferase reporter assays were outlined previously [ 23 , 24 , 25 , 26 ]. Briefly, a partial wild-type sequence, including the seed sequence of FOXD1 within the 3′-untranslated region (3′-UTR), was inserted into the psiCHECK-2 vector (C8021; Promega, Madison, WI, USA).…”

“…The procedures for immunohistochemistry were described in our previous studies [ 23 , 24 , 25 , 26 ]. The clinical samples were obtained from HNSCC cases who received surgical treatment at Chiba University Hospital.…”