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Transmembrane protein 40 (TMEM40) is a 23-kDa protein and its association with tongue squamous cell carcinoma (TSCC) remains unclear. This study aimed to investigate the expression and clinical significance of TMEM40 in TSCC and its roles in TSCC cells. Immunohistochemical analysis was performed to detect the expression levels of TMEM40 in 60 tongue tissue samples. Furthermore, TMEM40 was overexpressed and inhibited in two TSCC cell lines by transfection with pEZ-M98-TMEM40 plasmid or TMEM40 small interfering RNA, respectively. Cell Counting Kit-8 and colony formation assays were used to investigate the effects of TMEM40 on cell proliferation and colony formation ability, respectively. Flow cytometry was performed to determine cell apoptosis and cycle conditions of transfected cells. Wound-healing and Transwell assays were processed to explore the effects of TMEM40 on cell migration and invasion, respectively. The results indicated that TMEM40 expression levels were significantly increased in TSCC tissues compared with adjacent normal tongue tissues (P<0.01). Clinicopathological analysis revealed that TMEM40 expression was positively correlated with pathological TNM (pTNM) status (P<0.05), histological grade (P<0.001) and clinical stage (P<0.01), but not with sex or age. Results of cell proliferation, apoptosis, migration and invasion assays indicated that when TMEM40 had been successfully overexpressed or knocked down in CAL27 and SCC9 TSCC cell lines, cell growth and invasion increased in the TMEM40 overexpressing cells, while they decreased in TMEM40-knockdown cells. Furthermore, experiments revealed that TMEM40 knockdown resulted in increased levels of p53 and Bax, and decreased levels of MMP-9, which indicated that TMEM40 regulated cell apoptosis and migration via involvement of p53, Bax and MMP-9 in TSCC cells. Our findings indicated that increased expression of TMEM40 contributed to progressive features of TSCC via regulation of p53, Bax and MMP-9.

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