ABSTRACT
Small non-coding RNAs named microRNAs (miRNAs) modulate some functions and signaling pathways in skin epithelial cells and melanocytes. They also function as oncogenes or tumor suppressors in malignancies and tumor metastasis. We investigated the expression patterns of miRNAs, including miR-10b, 21, 200c, 373 and 520c, which regulate epithelial-to-mesenchymal transition (EMT) and metastasis in isolated cancer stem cells (CSCs) and non- CSCs. Six melanoma cell lines were tested for the expressions of stem cell markers. Melanoma stem cells were enriched via fluorescence-activated cell sorting (FACS) using the CD133 cell surface marker or spheroid culture. They were then characterized based on colony and sphere formation, and the expressions of stemness and EMT regulator genes and their invasion potential were assessed using real-time qRT-PCR and invasion assay. Our results indicate that cells enriched via sphere formation expressed all the stemness-related genes and had an enhanced number of colonies, spheres and invaded cells compared to cells enriched using the CD133 cell surface marker. Moreover, miRNAs controlling metastasis increased in the melanospheres. This may be related to the involvement of CSCs in the metastatic process. However, this must be further confirmed through the application of knockdown experiments. The results show that sphere formation is a useful method for enriching melanoma stem cells. Melanospheres were found to upregulate miR-10b, 21, 200c, 373 and 520c, so we suggest that they may control both metastasis and stemness potential.
Subject(s)
Epithelial-Mesenchymal Transition/genetics , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , MicroRNAs/genetics , Neoplastic Stem Cells/metabolism , AC133 Antigen , Antigens, CD/genetics , Antigens, CD/metabolism , Cell Line, Tumor , Cell Movement/genetics , Flow Cytometry , Gene Knockdown Techniques , Glycoproteins/genetics , Glycoproteins/metabolism , Humans , Melanoma/genetics , Melanoma/metabolism , Melanoma/pathology , Peptides/genetics , Peptides/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Spheroids, Cellular/metabolismABSTRACT
OBJECTIVE: MiR-302-367 is a cluster of polycistronic microRNAs that are exclusively expressed in embryonic stem (ES) cells. The miR-302-367 promoter is functional during embryonic development but is turned off in later stages. Motivated by the cancer stem cell hypothesis, we explored the potential expression of miR-302 in brain tumor cell lines. MATERIALS AND METHODS: In the present experimental study, we have tried to expand our knowledge on the expression pattern and functionality of miR302 cluster by quantifying its expression in a series of glioma (A-172, 1321N1, U87MG) and medulloblastoma (DAOY) cell lines. To further assess the functionality of miR-302 in these cell lines, we cloned its promoter core region upstream of the enhanced green fluorescent protein (EGFP) or luciferase encoding genes. RESULTS: Our data demonstrated a very low expression of miR-302 in glioma cell lines, compared with that of embryonal carcinoma cell line NT2 being used as a positive control. The expression of miR-302 promoter-EGFP construct in the aforementioned cell lines demonstrated GFP expression in a rare subpopulation of the cells. Serum deprivation led to the generation of tumorospheres, enrichment of miR-302 positive cells and upregulation of a number of pluripotency genes. CONCLUSION: Taken together, our data suggest that miR-302 could potentially be used as a novel putative cancer stem cell marker to identify and target cancer stem cells within tumor tissues.
ABSTRACT
PURPOSE: To investigate a potential alteration in the expression levels of transforming growth factor ß (TGF-ß) and miR-21 in bladder cancer tissues. MATERIAL AND METHODS: Using real-time polymerase chain reaction (PCR) method, we examined a potential correlated expression of miR-21 and TGF-ß variants in 30 bladder tumors and their marginal/non-tumor biopsy specimens obtained from the same patients. RESULTS: Our data revealed a significant down-regulation of TGF-ß variants (P = .03) along with a non-significant alteration in the expression of miR-21 in tumor vs. non-tumor samples. However, in contrast to low-grade tumors, the expression of miR-21 was upregulated in high-grade ones, and the expression level can efficiently discriminate low-grade tumors from high-grade ones (P = .03). CONCLUSION: In accordance to the observed similarity between TGF-ß variants and miR-21 gene expression alterations in bladder tumors, treating 5637 bladder cancer cell line with TGF-ß recombinant protein caused a significant upregulation of miR-21. The later finding further confirmed a correlated expression of TGF-ß and miR-21 in bladder tumors.
