Substrate Stiffness Drives Epithelial to Mesenchymal Transition and Proliferation through the NEAT1-Wnt/β-Catenin Pathway in Liver Cancer.

Xichao Xu,Shun Li,Yi Zhang,Liling Tang,Xing Wang

doi:10.3390/ijms222112066

Abstract

Background: Extracellular matrix (ECM)-derived mechanical stimuli regulate many cellular processes and phenotypes through mechanotransduction signaling pathways. Substrate stiffness changes cell phenotypes and promotes angiogenesis, epithelial to mesenchymal transition (EMT), and metastasis in tumors. Enhanced liver tissue matrix stiffness plays a crucial role in the tumorigenesis and malignant development of liver cancer and is associated with unfavorable survival outcomes. However, how liver cancer cells sense changes in ECM stiffness and the underlying molecular mechanisms are largely unknown. Methods: Seeding HepG2 cells on the micropillar gels, HepG2 cells were assessed for responsiveness to mechanotransduction using Western blot and immunofluorescence. Conclusions: We found that higher substrate stiffness dramatically enhanced malignant cell phenotypes and promoted G1/S transition in HepG2 cells. Furthermore, nuclear paraspeckle assembly transcript 1 (NEAT1) was identified as a matrix stiffness-responsive long non-coding RNA (lncRNA) regulating proliferation and EMT in response to increasing matrix stiffness during the progression of HepG2 cells towards liver cancer phenotypes. Higher matrix stiffness contributed to enhancing NEAT1 expression, which activated the WNT/β-catenin pathway. β-catenin translocates and enters the nucleus and the EMT transcription factor zinc finger E-box binding homeobox 1 (ZEB1) was upregulated to trigger EMT. Additionally, the proteins required for matrix stiffness-induced proliferation and resistance were strikingly upregulated in HepG2 cells. Therefore, our findings provide evidence that ECM-derived mechanical signals regulate cell proliferation and drive EMT through a NEAT1/WNT/β-catenin mechanotransduction pathway in the tumor microenvironment of liver cancer.

Highlights

Extracellular matrix (ECM) stiffness has been found to be involved in tumor metastasis and to drive epithelial to mesenchymal transition (EMT) by diverse mechanotransduction pathways [10,11]
These results confirmed that nuclear paraspeckle assembly transcript 1 (NEAT1) was upregulated in liver cancer and functioned as a substrate stiffness-associated long non-coding RNA (lncRNA) in HepG2 cells
Using a 5-ethynyl-2 -deoxyuridine (EdU) assay, we found that DNA synthesis ability was significantly enhanced after EdU treatment on a stiff substrate compared to a soft substrate, confirming that substrate stiffness could promote the proliferation of HepG2 cells (Figure 2A)

Summary

Introduction

Liver cancer is a main cause of cancer mortality globally. Along with chronic inflammation, fibrosis and cirrhosis can contribute to liver cancer initiation and progression [1]. High substrate stiffness promotes the expression of N-cadherin and vimentin, as well as increases the TGF-β1-induced Smad pathway in HCC [5] These findings suggest a role for ECM stiffness in triggering EMT during cancer metastasis. How ECM stiffness impacts liver cancer cellular processes and modulates liver cancer survival and metastasis and the underlying molecular mechanism are largely unknown. Extracellular matrix (ECM)-derived mechanical stimuli regulate many cellular processes and phenotypes through mechanotransduction signaling pathways. Nuclear paraspeckle assembly transcript 1 (NEAT1) was identified as a matrix stiffness-responsive long non-coding RNA (lncRNA) regulating proliferation and EMT in response to increasing matrix stiffness during the progression of HepG2 cells towards liver cancer phenotypes. Our findings provide evidence that ECM-derived mechanical signals regulate cell proliferation and drive EMT through a NEAT1/WNT/β-catenin mechanotransduction pathway in the tumor microenvironment of liver cancer

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