Micro-RNAs (miRs) are emerging as important players in carcinogenesis. Their stromal expression has been less investigated in part due to lack of methods to accurately differentiate between tumor compartments. This study aimed to establish a robust method for dual visualization of miR and protein (pan-cytokeratin) by combining chromogen-based in situ hybridization (ISH) and immunohistochemistry (IHC), and to apply it to investigate stromal expression of miR204 as a putative prognostic biomarker in oral squamous cell carcinoma (OSCC). Four different combinations of methods were tested and ImageJ and Aperio ImageScope were used to quantify miR expression. All four dual ISH-IHC methods tested were comparable to single ISH in terms of positive pixel area percentage or integrated optical density of miRs staining. Based on technical simplicity, one of the methods was chosen for further investigation of miR204 on a cohort of human papilloma virus (HPV)-negative primary OSCC (n = 169). MiR204 stromal expression at tumor front predicted recurrence-free survival (p = 0.032) and overall survival (p = 0.036). Multivariate Cox regression further confirmed it as an independent prognostic biomarker in OSCC. This study provides a methodological platform for integrative biomarker studies based on simultaneous detection and quantification of miRs and/or protein and reveals stromal miR204 as a prognostic biomarker in OSCC.

Background: Knowledge on the role of miR changes in tumor stroma for cancer progression is limited. This study aimed to investigate the role of miR dysregulation in cancer-associated fibroblasts (CAFs) in oral squamous cell carcinoma (OSCC). Methodology: CAF and normal oral fibroblasts (NOFs) were isolated from biopsies of OSCC patients and healthy individuals after informed consent and grown in 3D collagen gels. Total RNA was extracted. Global miR expression was profiled using Illumina version 2 panels. The functional impact of altered miR-204 expression in fibroblasts on their phenotype and molecular profile was investigated using mimics and inhibitors of miR-204. Further, the impact of miR-204 expression in fibroblasts on invasion of adjacent OSCC cells was assessed in 3D-organotypic co-cultures. Results: Unsupervised hierarchical clustering for global miR expression resulted in separate clusters for CAF and NOF. SAM analysis identified differential expression of twelve miRs between CAF and NOF. Modulation of miR-204 expression did not affect fibroblast cell proliferation, but resulted in changes in the motility phenotype, expression of various motility-related molecules, and invasion of the adjacent OSCC cells. 3′ UTR miR target reporter assay showed ITGA11 to be a direct target of miR-204. Conclusions: This study identifies differentially expressed miRs in stromal fibroblasts of OSCC lesions compared with normal oral mucosa and it reveals that one of the significantly downregulated miRs in CAF, miR-204, has a tumor-suppressive function through inhibition of fibroblast migration by modulating the expression of several different molecules in addition to directly targeting ITGA11.

The majority of severely ill patients experience dry mouth. For institutionalized patients, this condition is commonly treated using glycerol as a lubricant. However, because of its possibly desiccating effect, some countries do not advocate the use of glycerol. This study aimed to investigate dose‐dependent effects of glycerol on homeostasis and tissue integrity of in vitro‐reconstructed normal human buccal mucosa (RNHBM). Primary keratinocytes and fibroblasts were isolated and expanded from biopsies of mucosa from eight healthy volunteers. Ninety‐six samples of RNHBM were prepared and exposed for 24 h to 17%, 42.5%, or 85% glycerol, or to distilled H2O (control). Sections were stained with haematoxylin and eosin (H&E) to evaluate epithelial thickness or used for immunohistochemistry to measure expression of Ki67 (proliferation), cleaved caspase‐3 (apoptosis), and E‐cadherin (tissue‐integrity). Positive cells and cell layers, as detected by immunohistochemistry, were counted. Epithelial thickness, proliferation, and apoptosis were significantly increased by exposure to 42.5% and 85% glycerol. No significant differences in apoptosis or proliferation were found between controls and RNHBM exposed to 17% glycerol. E‐cadherin expression was not significantly affected by exposure to any of the concentrations of glycerol tested. This study shows that glycerol affects tissue homeostasis, but not tissue integrity, of RNHBM at glycerol concentrations above 42.5%.

BackgroundRecent studies have shown aberrant expression of micro-RNAs in cancer-associated fibroblasts (CAFs). This study aimed to investigate miR-138 dysregulation in CAFs in oral squamous cell carcinoma (OSCC) and its effects on their phenotype and invasion of adjacent OSCC cells.MethodsExpression of miR-138 was first investigated in OSCC lesions (n = 53) and OSCC-derived CAFs (n = 15). MiR-138 mimics and inhibitors were used to functionally investigate the role of miR-138 on CAF phenotype and the resulting change in their ability to support OSCC invasion.ResultsExpression of miR-138 showed marked heterogeneity in both OSCC tissues and cultured fibroblasts. Ectopic miR-138 expression reduced fibroblasts’ motility and collagen contraction ability and suppressed invasion of suprajacent OSCC cells, while its inhibition resulted in the opposite outcome. Transcript and protein examination after modulation of miR-138 expression showed changes in CAF phenotype-specific molecules, focal adhesion kinase axis, and TGFβ1 signaling pathway.ConclusionsDespite its heterogeneous expression, miR-138 in OSCC-derived CAFs exhibits a tumor-suppressive function.