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

Sep 2, 2015International journal of oncology

MicroRNA-34a controls cell changes and cancer stem traits in head and neck squamous cell carcinoma cells

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Abstract

miR-34a expression levels were significantly downregulated in the majority of spheroid-derived cells from head and neck squamous cell carcinoma (HNSCC), showing a decrease of 1.6-16.4-fold compared to parental monolayer-derived cells.

Spheroid-derived cells (SDC) from HNSCC lines exhibited significantly increased expression of epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) related transcription factors (TFs) compared to parental monolayer-derived cells (MDC), with levels rising up to 36.8-fold.
ALDH expression was significantly greater in SDC, with an increase of 2-3-fold.
HPV-negative HNSCC lines demonstrated higher mean expression levels of EMT-TFs, CSC-TFs, and ALDH compared to HPV-positive lines (30.3% vs. 12.8%).
Transfection of miR-34a mimics led to a significant reduction in the expression levels of EMT- and CSC-related TFs in both HPV-positive and HPV-negative SDC.
The invasive capacity and clonogenic ability of HNSCC-SDC were inhibited by miR-34a mimics, with reductions of up to 30% in invasiveness.

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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 HPV- group 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.

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

Abstract

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