Background The objective of this study was to clarify the molecular mechanism of amoeboid‐to‐mesenchymal transition (AMT) of CD44high oral squamous cell carcinoma (OSCC) cells. Methods Morphology and expression of mesenchymal genes were investigated in CD44high OSCC cells (CD44high OM‐1 cells) cultured on laminin‐coated soft silicone gel. Additionally, microarray analysis was performed to investigate microRNA (miRNA) expression inhibited by transforming growth factor‐β1 (TGF‐β1) in CD44high OM‐1 cells. Results When CD44high OM‐1 cells were cultured on 2.0‐kPa laminin‐coated silicone gel, the cells exhibited an amoeboid‐like round morphology. Cofilin‐1 expression was found in the nucleus and cytoplasm of amoeboid‐like CD44high OM‐1 cells. The invasive capacity was significantly reduced after Cofilin‐1 knockdown. Additionally, Cofilin‐1 knockdown cells had an irregularly extended shape. Phosphorylated Cofilin‐1 was significantly upregulated by TGF‐β1. Additionally, TGF‐β1 enhanced N‐cadherin and Snail mRNA expression and induced a spindle‐shaped morphology. ERK1/2 phosphorylation was induced by TGF‐β1. Microarray analysis revealed that miR‐422a exhibited the greatest downregulation (fold change: 0.22) in the presence of TGF‐β1. Importantly, TGF‐β1‐inhibited miR‐422a expression was recovered by the ERK inhibitor or ERK1/2 knockdown. Additionally, miR‐422a inhibitor‐transfected CD44high OM‐1 cells exhibited high N‐cadherin and Snail mRNA expression. Furthermore, Cofilin‐1 knockdown and miR‐422a inhibition induced a spindle cell morphology. Conclusion Cofilin‐1 is involved in the invasive ability of CD44high OSCC cells. TGF‐β1 contributes to AMT by downregulation of miR‐422a via ERK activation and Cofilin‐1 phosphorylation. Our findings suggest that miR‐422a and Cofilin‐1 play major roles in the maintenance of amoeboid‐like CD44high cells.

our previous study revealed that treatment with a combination of fibroblast growth factor-2 and melatonin (MEL) synergistically augmented osteogenic activity and mineralization of Mc3T3-e1 mouse preosteoblast cells. Thus, the objective of the present study was to assess the effect of MEL on osteogenetic characteristics in human osteoblastic cells. Human jawbone-derived osteoblastic (hoB) cells were isolated from mandibular bone fragments. RUNX family transcription factor 2 (runx2) expression, alkaline phosphatase (alP) enzyme activity and the mineralization ability of hoB cells in the presence of Mel were evaluated. Microarray analysis was also performed to assess the expression of Mel-induced micrornas (mirnas/mirs) in hOB cells. Treatment with MEL significantly enhanced runx2 expression, alP activity and mineralization staining. However, this effect was significantly reduced following transforming growth factor-β1 treatment. in total, 124 mirnas were differentially expressed in Mel-treated hOB cells, compared with untreated cells. Of the upregulated miRNAs, miR-181c-5p exhibited the largest fold change. Runx2 mRNA expression and mineralization staining in the presence of MEL were significantly reduced following transfection with a mir-181c-5p inhibitor. in addition, transfection with miR-181c-5p mimics significantly increased Runx2 expression and mineralization staining. These results suggested that MEL-induced miR-181c-5p was involved in osteogenic differentiation and mineralization of hOB cells. Using TargetScan, a putative miR-181c-5p binding site was identified in the Notch2 gene. Moreover, Notch2 mRNA and protein expression levels in hOB cells were significantly reduced following transfection with miR-181c-5p mimics, confirming Notch2 as a target gene for miR-181c-5p. Notch2 siRNA knockdown significantly increased Runx2 expression and mineralization staining, which suggested that Notch2 may negatively regulate osteogenic differentiation of hOB cells by downregulating Runx2. In conclusion, MEL-induced expression of miR-181c-5p enhanced osteogenic differentiation and calcification of hOB cells.

We previously found that microRNAs play major roles in the maintenance of amoeboid-like oral squamous cell carcinoma (OSCC) cells with high expression of CD44 (CD44 high ). However, the roles of microRNAs in chemotherapeutic resistance exhibited by CD44 high amoeboid-like OSCC cells are unclear. Here, docetaxelinduced apoptosis was examined in CD44 high OSCC cells (CD44 high OM-1 cells) cultured on laminin-coated silicone gel. Amoeboid-like CD44 high OSCC cells exhibited robust resistance to docetaxel-induced apoptosis and significant upregulation of miR-224-5p expression compared with epithelial-like CD44 high OSCC cells and mesenchymal-like CD44 high OSCC cells. The expression of pannexin-1 (PANX1), a channel-forming protein that regulates the release of ATP, was significantly upregulated following transfection of amoeboid-like CD44 high OSCC cells with an miR-224-5p inhibitor. These results suggest that miR-224-5p inhibits PANX1 expression. Furthermore, miR-224-5p inhibitor-transfected amoeboid-like CD44 high OSCC cells exhibited significant enhancement of the proportion of apoptotic cells; however, this effect was significantly inhibited by knockdown of PANX1 with PANX1 small interfering RNA. Additionally, the miR-224-5p inhibitor-enhanced extracellular ATP levels were significantly reduced by PANX1 knockdown. These findings imply that miR-224-5p plays a vital role in the resistance to docetaxel-induced apoptosis by attenuating PANX1-induced ATP discharge. Moreover, amoeboid-like CD44 high OSCC cells may be involved in chemotherapeutic resistance of OSCC.

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Previous studies have demonstrated that oral Epstein-Barr virus (EBV) is associated with periodontitis. However, the relationship between periodontitis and oral EBV has not been fully elucidated by reducing the effects of confounding factors. The aim of the present study was to clarify the association between oral Epstein-Barr virus (EBV) and oral health status among middle-aged and older Japanese individuals. A total of 124 patients (46 males and 78 females; mean age, 69.2 years; age range, 35-90 years) who visited Hiroshima University Hospital between October 2018 and December 2019 were recruited into the present study. EBV DNA positivity was determined in 124 oral rinse samples using quantitative PCR. Periodontal disease-related bacteria were also detected by PCR analysis. EBV DNA was determined as positive in 16 of the 124 enrolled patients (12.9%). No significant difference was identified between EBV DNA and clinical factors (sex, age, remaining teeth, denture use, smoking or medical history). Of the 38 patients with periodontal pockets ≥6 mm, 10 were EBV DNA positive (26.3%). There was a significant association between EBV DNA positivity and probing depth (P=0.01). Additionally, a significant association was identified between bleeding on probing (BOP) and EBV DNA positivity (P=0.03). To investigate the relationship between EBV and periodontal health status, propensity score-matching was determined between participants without ≥4 mm periodontal pockets and BOP (participants with good periodontal health) and those with ≥4 mm periodontal pockets, BOP or both (participants with poor periodontal health). A total of 35 matched pairs were identified among the patients. Patients with poor periodontal health exhibited a higher EBV DNA positivity rate (25.7%) than those with good periodontal health (0.0%). Additionally, there was a significant association between EBV DNA positivity and periodontal health status (P=0.001). T. denticola-positive participants exhibited a higher EBV DNA positivity rate than negative participants (17.6 vs. 9.6%). However, there was no significant difference. The results indicated that oral EBV may be markedly associated with periodontitis in middle-aged and older Japanese individuals.