Involvement of the A disintegrin and metalloproteinase 9 in oral cancer cell invasion

Abstract

The aims of this study were to determine the functional roles of the transmembrane glycoprotein, Disintegrin and metalloproteinase domain-containing protein 9 (ADAM 9), in the phosphorylation of epidermal growth factor receptor (EGFR) and AKT and in the aggressiveness of oral cancer cells. Immunohistochemistry and immunoblotting were conducted to determine expression of ADAM 9 and the levels of EGFR phosphorylated at the tyrosine 1173 residue (p-EGFR tyr1173 ) and AKT phosphorylated at the serine 473 residue (p-AKT ser473 ) in oral cancer tissues and in the oral cancer cell lines HN5, HN6, HN15, and HN008. Small interfering RNA (siRNA) was used to inhibit expression of ADAM9 mRNA, and thus production of ADAM9 protein, in oral cancer cells. ADAM9-knockdown cells were examined for p-EGFR tyr1173 and p-AKT ser473 levels and used for cell proliferation and invasion assays. A positive correlation among overexpression of ADAM 9, p-EGFR tyr1173 , and p-AKT ser473 was found in oral cancer tissues.

Overexpression of methyltransferase‐like 3 and 14 in oral squamous cell carcinoma

“…As previously demonstrated, 18 transfection with siADAM9 in HN6 significantly decreased cell invasion (p < 0.001; Figure 3E). Transfection with siMETTL3, siMETTL14, or both in HN6 also failed to inhibit cell migration for 72 h (Figure 3F and G).…”

“…Three cell lines derived from patients with OSCC, including HN5, HN6, HN15, and five different cell lines of normal human oral keratinocytes (HOKs), retrieved from our previous study, 18 were grown in serum-free keratinocyte growth medium (KGM; BioWhittaker ® ), at 37℃ in a humidified atmosphere with 5% CO 2 . KGM was replenished every two days until cell growth reached 80% confluence.…”

“…18 The reason to include ADAM9 as a positive control in cell transfection was due to the results of our recently published work that demonstrated its involvement in HN6 invasion. 18 Then, the transfection medium was replaced with serum-free KGM for 72 h. Subsequently, an assessment for the efficiency of transfection by analyzing mRNA and protein expressions of METTL3, METTL14, and ADAM9, as well as analyses of cell viability by MTT assay and of cancer cell aggressiveness by BrdU and Transwell chamber assays in transfected HN6, were compared with the non-transfected HN6. For the wound healing assay, both transfected and non-transfected HN6 were observed every 1 h during the 72-h incubation in serum-free KGM.…”

“…A 2µg quantity of total RNA was converted into complementary DNA (cDNA) using the RevertAid First Strand cDNA Synthesis kit (ThermoFisher Scientific). qPCR was performed using 5% of cDNA and the SYBR No-ROX kit (SensiFAST™, Bioline), containing Taq polymerase, SYBR green, and a specific primer pair (10 μM each) for METTL3, METTL14, β-catenin, ADAM9, 16,18 as positive controls for aberrant expressions in OSCC, or glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as a housekeeping gene, by the LightCycler 480 (Roche). The primers' sequences are summarized in Table S1.…”

“…OSCC cells and HOKs as well as transfected and non-transfected HN6 were lysed using radio-immunoprecipitation assay buffer and their total protein content was determined, as previously described. 18 A 40μg quantity of total protein was resolved on 10%…”