Research Article: TNF-α-induced miR-450a mediates TMEM182 expression to promote oral squamous cell carcinoma motility

Date Published: March 20, 2019

Publisher: Public Library of Science

Author(s): En-Wei Hsing, Shine-Gwo Shiah, Hsuan-Yu Peng, Ya-Wen Chen, Chih-Pin Chuu, Jenn-Ren Hsiao, Ping-Chiang Lyu, Jang-Yang Chang, Aamir Ahmad.


Distant metastasis leads oral cancer patients into a poor survival rate and a high recurrence stage. During tumor progression, dysregulated microRNAs (miRNAs) have been reported to involve tumor initiation and modulate oral cancer malignancy. MiR-450a was significantly upregulated in oral squamous cell carcinoma (OSCC) patients without functional reports. This study was attempted to uncover the molecular mechanism of novel miR-450a in OSCC. Mir-450a expression was examined by quantitative RT-PCR, both in OSCC cell lines and patients. Specific target of miR-450a was determined by software prediction, luciferase reporter assay, and correlation with target protein expression. The functions of miR-450a and TMEM182 were accessed by adhesion and transwell invasion analyses. Determination of the expression and cellular localization of TMEM182 was examined by RT-PCR and by immunofluorescence staining. The signaling pathways involved in regulation of miR-450a were investigated using the kinase inhibitors. Overexpression of miR-450a in OSCC cells impaired cell adhesion ability and induced invasiveness, which demonstrated the functional role of miR-450a as an onco-miRNA. Interestingly, tumor necrosis factor alpha (TNF-α)-mediated expression of TMEM182 was regulated by miR-450a induction. MiR-450a-reduced cellular adhesion was abolished by TMEM182 restoration. Furthermore, the oncogenic activity of TNF-α/miR-450a/TMEM182 axis was primarily through activating extracellular signal–regulated kinase 1/2 (ERK1/2) signaling pathway. ERK1/2 inhibitor prevented the TNF-α-induced miR-450a expression and enhanced adhesion ability. Our data suggested that TNF-α-induced ERK1/2-dependent miR-450a against TMEM182 expression exerted a great influence on increasing OSCC motility. Overall, our results provide novel molecular insights into how TNF-α contributes to oral carcinogenesis through miR-450a that targets TMEM182.

Partial Text

Oral squamous cell carcinoma (OSCC) is the most lethal type in head and neck squamous cell carcinoma (HNSCC) in the world. Over the past decades, the incidence rate of OSCC has increased among younger generations [1–3]. In spite of considerable advances in surgery, radiotherapy and chemotherapy, the 5-year survival rate for OSCC has not improved markedly because patients still frequently arise loco-regional recurrence and lymph node metastasis [4–6]. Hence, finding new biomarker(s) and therapeutic molecule(s) is urgent. Recently, a growing evidence indicates that microRNAs (miRNAs) contribute to the initiation and development of oral cancer [7–9]. Therefore, exploring unique miRNAs and related molecular pathways underlying OSCC aggressive will provide advantages to improve therapeutic efficacy, as well as to design more effective treatment strategies.

MiR-450a is identified as one of prognostic markers for adrenocortical tumor, aristolochic acid nephropathy and type 2 diabetes [23–25]. Emerging data indicated that miR-450a controls a divergent function on the osteoblastic differentiation of human mesenchymal stem cells by targeting STAT1 and rats’ adipogenesis by targeting WISP2 [26, 27]. However, there are no data to suggest a connection between miR-450a and oral cancer in recent investigations. In this study, gain-of function approach indicated that miR-450a restoration significantly inhibited cell adhesion and enhanced invasion ability in DOK and SAS cells, suggesting miR-450a is an onco-miR and play an important role in OSCC cellular adhesion on matrix. For elucidation the molecular mechanisms and identification the putative targets of miR-450a in OSCC, we performed genome-wide gene expression analysis using miR-450a transfected DOK and SAS cells. In silico analysis the expression signatures of miR-450a transfectants in OSCC cells and OSCC patients, we found that twelve genes were downregulation in clinical specimens and that they were candidate targets of miR-450a. Among these genes, transmembrane protein TMEM182 showed a best inverse correlation with miR-450a. Downregulating TMEM182 using shRNA suppressed cell adhesion in a manner comparable to miR-450a overexpression. Moreover, the restoration of TMEM182 potently rescued the adhesion ability suppressed by miR-450a, suggesting that miR-450a mainly acts as a novel regulator in mediating OSCC adhesion by targeting membrane protein TMEM182.