HOTAIR Promotes the Hyperactivation of PI3K/Akt and Wnt/β-Catenin Signaling Pathways via PTEN Hypermethylation in Cervical Cancer

Abstract

The mechanisms underlying the sustained activation of the PI3K/AKT and Wnt/β-catenin pathways mediated by HOTAIR in cervical cancer (CC) have not been extensively described. To address this knowledge gap in the literature, we explored the interactions between these pathways by driving HOTAIR expression levels in HeLa cells. Our findings reveal that HOTAIR is a key regulator in sustaining the activation of both signaling pathways. Specifically, altering HOTAIR expression-either by knockdown or overexpression-significantly influenced the transcriptional activity of the PI3K/AKT and Wnt/β-catenin pathways. Additionally, we discovered that HIF1α directly induces HOTAIR transcription, which in turn leads to the epigenetic silencing of the PTEN promoter via DNMT1. This process leads to the sustained activation of both pathways, highlighting a novel regulatory axis involving HOTAIR and HIF1α in cervical cancer. Our results suggest a new model in which HOTAIR sustains reciprocal activation of the PI3K/AKT and Wnt/β-catenin pathways through the HOTAIR/HIF1α axis, thereby contributing to the oncogenic phenotype of cervical cancer.

Keywords: HOTAIR; LncRNAs; PI3K/AKT pathway; Wntl/β-catenin pathway; epigenetic regulation.

Figure 1

Enrichment of HOTAIR-associated transcription factors and pathways. (A) Enrichment of HOTAIR-associated signaling pathways from Webgestalt. (B) Enrichment of transcription factors associated with HOTAIR from Shinygo. (C) HOTAIR expression in CC cell lines in comparison with non-tumoral cell line. (D) Interaction probability of HOTAIR with β-catenin and HIF1α and its experimental corroboration by RIP assay. p < 0.05 (*). p < 0.01 (**). p < 0.001 (***).

Figure 2

PI3K/AKT regulates HOTAIR expression and Wnt/β-catenin activation in HeLa cell line. (A) IC50 and Western blot of PI3K/AKT pathway with BKM120 inhibitor. (B) BKM120 inhibits transcriptional activity of HIF1α, determined by measuring luciferase reporter and Glut1 and HK2 expression in HeLa cells. (C) HOTAIR levels detected in HeLa cells treated with anti-sense probe and BKM120 inhibitor. Bar = 30 and 10 μm. (D) Inhibition of HOTAIR expression with BKM inhibitor treatment. (E) Wnt/β-catenin transcriptional activity evaluated by TOP Flash activity and Cyclin D1, c-Myc expression with BKM120 inhibitor treatment. p < 0.05 (*). p < 0.01 (**). p < 0.001 (***).

Figure 3

HOTAIR regulates the Wnt/β-catenin and PI3K/AKT pathways in the HeLa cell line. (A) Expression levels of HOTAIR by different amounts of anti-sense probe. (B) HOTAIR overexpression in HeLa cell line. (C) Inhibition of HOTAIR affects transcriptional activity of HIF1α by luciferase activity, and HIF1α targets Glut1 and HK2 expression. (D) HOTAIR overexpression increases HIF1α transcriptional activity by luciferase activity, and HIF1α targets expression. (E) HOTAIR knockdown decreases Wnt/β-catenin transcriptional activity, as evaluated by TOP Flash activity and Cyclin D1, c-Myc expression. (F) HOTAIR overexpression increases Wnt/β-catenin transcriptional activity, as evaluated by TOP Flash activity and Cyclin D1, c-Myc. p < 0.05 (*). p < 0.01 (**). p < 0.001 (***).

Figure 4

HIF1α-mediated expression of HOTAIR in the HeLa cell line (A) HOTAIR promoter is enriched with Wnt response elements (WRE) and hypoxia response elements (HRE). (B,C) Stabilization of HIF1α by DMOG and assessment of its transcriptional activity in the HeLa cell line. (D) Over-activation of the transcriptional activity of Wnt/β- catenin by LiCl in HeLa cell line. (E,F) HOTAIR expression upon over-activation of HIF1α and Wnt/β-catenin pathway transcriptional activity by LiCl and DMOG in HeLa cell line. (G) HIF-1α is located in HOTAIR promoter by ChIP assay. p < 0.05 (*). p < 0.01 (**). p < 0.001 (***).

Figure 5

HOTAIR-mediated expression of PTEN. (A) PTEN expression levels in HeLa cell line. (B) PTEN expression upon HOTAIR inhibition and overexpression evaluated by qPCR and Western blot. (C) DNMT1 expression levels in HeLa cell line. (D) Interaction probability of HOTAIR with DNMT1 and its experimental corroboration by RIP assay. (E) 5-mC IP of PTEN promoter upon HOTAIR inhibition and overexpression. p < 0.05 (*). p < 0.01 (**). p < 0.001 (***).

Figure 6

HOTAIR-mediated over-activation of PI3K/AKT and Wnt/β-catenin signaling pathways through nuclear processes, methylation of PTEN promoter and feedback with HIF1α.