Abstract
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.
Highlights
In a recent study on a cohort of 169 patients with human papilloma virus (HPV)-negative primary oral squamous cell carcinoma (OSCC), we showed that presence of miR-204 in the stroma at the tumor front predicted better overall survival and
Among the twelve dysregulated miRNA identified in our study, we focused further on miR-204 in functional and molecular assays owing to a possible link that we found in silico between this particular miRNA and integrin alpha11, which has been identified as one of the top genes significantly up-regulated in cancer-associated fibroblasts (CAFs) derived from OSCC in our previous transcriptomic study [19]
We show in this study that several other CAF-related molecules were modulated by miR-204, endorsing a more complex role of miR-204 in regulating the CAF phenotype than only through integrin alpha 11 (ITGA11)
Summary
Oral cancer represents a significant proportion of head and neck cancer and is an important cause of morbidity and mortality worldwide [1]. The discovery of micro-RNAs (miRs) as regulators of gene expression [9–11], and evidence of their importance for development and disease [12], has led to a burst of miR studies in cancers, including OSCC. This study aimed to investigate the role of miR dysregulation in cancer-associated fibroblasts (CAFs) in oral squamous cell carcinoma (OSCC). 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. 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
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