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Evaluation of miRNAs expression that regulates the ZEB1, involved in epithelial-mesenchymal transition in head and neck cancer stem cells


Introduction: The Head and neck squamous cell carcinoma (HNSCC) is the sixth most common neoplasm in the world. Despite considerable advances in therapy, the late diagnosis associated to a secondary tumor formation results in a survival rate of only 35% at five years. These data show that new strategies are needed for improving early diagnosis, predicting prognosis, and establishing effective therapeutics. One of the possibilities is the identification of biomarkers such as cancer stem cells (CSC) which have the potential of self-renewal and differentiation. Evidence proved that the CSC act in tumor initiation, progression, metastasis, and radio and chemo-resistance. Another biomarker that is being extensively studied is the micro-RNA (miRNA), small non-coding RNAs. These establish the post-transcriptional regulation of gene expression of target genes, leading to inhibition or overexpression of the proteins. The miRNAs regulate cellular proliferation, differentiation, migration, apoptosis, survival, morphogenesis, and carcinogenesis. Altered expression of miRNAs can lead to inhibition of tumor suppressor genes or to oncogene overexpression, as well as the development of cancer. Recent studies have also shown that miRNAs regulate CSC. However, the expression profile and the role of miRNAs on these cells are yet unknown. Objectives: Assess the miRNAs expression that regulates ZEB1 (zinc finger E-box binding homeobox 1), and that is involved in the epithelial-mesenchymal transition (EMT), in CSC and in non-stem cancer cell (non-CSC) isolated from HNSCC. The specific objectives of this project are: 1) assess the miRNAs (miR-101, miR-139, miR-153, mir-200 e mir-205) expression, that regulates ZEB1, and are involved in EMT, in CSC CD44+/ALDH1+ and in non-CSC ALDH1-/CD44-, which were obtained from primary cultures of HNSCC; 2) compare the expression level of the miRNAs of the CD44+/ALDH1+ ALDH1-/CD44- cells, in an attempt to identify differences between the two types of cells that may be related to tumor aggressiveness; 3) perform the functional analysis, in vitro, of the two miRNAs that present greater difference in expression to confirm if they really regulate the ZEB1. Methods: It will be evaluated the miRNAs expression, predicted for ZEB1, in five samples of the HNSCC. The samples will be subject to primary culture for subsequent isolation of the CSC by flow cytometry, using CD44 and ALDH1 markers. It will be assessed the tumorigenic potential of ALDH1+/CD44+ and ALDH1-/CD44- cells by their ability in generate tumor spheres. Their capability of invasion and migration in Matrigel chamber will also be analyzed. After these steps, the cells will be multiplied and subjected to extraction of total RNA for expression analysis of miRNAs predicted for ZEB1, by RT-qPCR. Two samples that show more differentially patterns of miRNAs expression will be select for functional analysis. For this analysis will be used the transfection technique miRNA mimic, followed by analysis of the miRNAs expression by RT-qPCR. It will also be evaluated the gene and protein expression of ZEB1 by RT-qPCR and Western Blot, respectively. All results will be evaluated by appropriate statistical analysis. Expected results: It is expected that the present study can contribute to define the miRNA expression of CSC of HNSCC and thus provide data for description of a possible molecular biomarker for HNSCC, which can be use as therapeutic targets for new drugs. (AU)