MicroRNA-34a regulates epithelial-mesenchymal transition and cancer stem cell phenotype of head and neck squamous cell carcinoma in vitro

Abstract

MicroRNAs (miRs) are short non-coding single stranded RNAs regulating the translation of target mRNAs in normal and cancer cells in which they are frequently dysregulated promoting tumor progression. Cancer stem cells (CSCs) of head and neck squamous cell carcinoma (HNSCC), identified by aldehyde-dehydrogenase expression (ALDH), are a cell subset within the tumor cell population that takes part in the genesis and progression of cancer. The relevance of epithelial-mesenchymal transition (EMT) has recently been recognized for tumor development and metastasis. Several studies have illustrated that miRs regulate EMT of CSC. CSC from 8 HNSCC lines, 4 of which are human papillomavirus (HPV)-positive, were enriched by spheroid culture (spheroid-derived cells, SDC) and compared to their parental monolayer-derived cells (MDC) to analyze expression patterns of miR-34a, CSC-related transcription factors (CSC-TFs: Sox2, Nanog, Oct3/4) and EMT-related TFs (EMT-TFs: Twist, Snail1, Snail2) by RT-qPCR. Flow cytometry was used to quantify and enrich ALDH+ CSCs. Transfection of miR-34a mimics was used to evaluate its regulatory potential for CSC marker profiles as well as CSC- and EMT-TFs expression in HNSCC-SDC. Invasive, colony-forming and clonogenic capability of the miR-34a mimics transfected SDC after sorting for ALDH+ and ALDH- cells was assessed by Matrigel invasion, clonogenicity and spheroid formation assay, respectively. miR-34a expression levels were significantly downregulated in the majority of SDC derived from HNSCC-lines as compared to parental MDC (-1.6-16.4-fold). For EMT- and CSC-related TF expression, all HNSCC-derived SDC showed a significantly increased level compared to parental MDC (≤36.8-fold). Significantly increased expression of ALDH was found in SDC (2-3-fold). Compared to the HPV+, the HPVgroup showed a significantly higher mean expression level of EMT-TFs, CSCs-TFs and ALDH (30.3 v.s. 12.8%). Transfection of miR-34a mimics significantly reduced the EMT- and CSC-related TF expression level in UM-SCC9 (HPV-) and UM-SCC47 (HPV+) SDC. Simultaneously, the ALDH expression was reduced significantly (1.5-2-fold) and the invasive capacity (≤30%) and clonogenicity of HNSCC-SDC was also inhibited by transfection of miR-34a mimics compared to controls. Restoration of miR-34a significantly inhibited the capability for EMT formation of CSC-phenotype and functionally reduced clonogenic and invasive capacity in HNSCC cell lines. Therapeutic modulation of miR-34a in HNSCC and CSCs may reduce the rate of metastasis and recurrence of tumors after therapy.