HOTAIR Knockdown Decreased the Activity Wnt/β-Catenin Signaling Pathway and Increased the mRNA Levels of Its Negative Regulators in Hela Cells
Cellular Physiology and Biochemistry
HOTAIR is a long non-coding RNA that promotes the development of human cancer. TET1 enzyme is involved in DNA demethylation by oxidation of 5-methylcytocine and it is considered a tumor suppressor in some types of cancer. HOTAIR and TET1 are involved in modulation of the Wnt/β-catenin signaling pathway, but their role in cervical cancer remains to be elucidated. The aim of this work was to analyze the effect of HOTAIR in TET1 expression, Wnt/β-catenin signaling, and expression, methylation and
... n, methylation and hidroxymethylation of some negative regulators of this pathway in HeLa cells. HOTAIR and TET expression were analyzed by RT-qPCR and western blot. The HOTAIR knockdown was done with DsiRNA and the activity of the Wnt/β-catenin signaling pathway through luciferase assays and β-catenin nuclear translocation. The mRNA levels of SNAIL, EDN3, CYCD1, SPRY2 (targets of Wnt/β-catenin pathway) PCDH10, SOX17, AJAP1, and MAGI2 (negative regulators of Wnt/β-catenin pathway) were evaluated by RT-qPCR. The DNA methylation and hidroxymethylation of negative regulators of the Wnt/β-catenin pathway were evaluated by methylation-specific PCR and chemical modification, followed by digestion and quantitative PCR. HOTAIR knockdown in HeLa cells decreased the activity of Wnt/β-catenin signaling pathway. It increased the mRNA levels of Wnt/ β-catenin negative regulators through a decrease in their promoter's methylation pattern. TET1 enzyme was also down-regulated in HOTAIR knockdown cells. Our study suggests a mechanism in which HOTAIR promotes the over-activation of Wnt/β-catenin signaling pathway by downregulation of PCDH10, SOX17, AJAP1 and MAGI2 and also TET.