The silencing of LncRNA-H19 decreases chemoresistance of human glioma cells to temozolomide by suppressing epithelial-mesenchymal transition via the Wnt/β-Catenin pathway

Abstract

Introduction: Temozolomide (TMZ) is commonly used for glioma chemotherapy. However, TMZ resistance limits the therapeutic effect of TMZ in glioma treatment. LncRNA-H19 acts as an oncogenic LncRNA in some types of cancers and has been reported to be up-regulated in glioma.

Materials and methods: In our present study, we established TMZ-resistant glioma cells (U-251TMZ and M059JTMZ) to explore the effect of H19 on the chemoresistance of glioma cells.

Results: We observed that the expression of H19 was significantly increased in U-251TMZ and M059JTMZ cells. Knockdown of H19 expression using specific shRNA in U-251TMZ and M059JTMZ led to decreased half maximal inhibitory concentration (IC₅₀) values for TMZ and increased cell apoptosis rates, indicating that the silencing of H19 decreased chemoresistance of glioma cells to TMZ. In addition, silencing of H19 suppressed epithelial-mesenchymal transition (EMT) by increasing the expression of epithelial marker E-cadherin and decreasing the expression of mesenchymal marker Vimentin and ZEB1. Moreover, inducing EMT by TGF-β1 treatment led to increased IC₅₀ values for TMZ and decreased cell apoptosis rates compared with TMZ+H19 shRNA group, suggesting that the induction of EMT counteracted the inhibitory effect of H19 shRNA on chemoresistance of glioma cells to TMZ. Furthermore, the reduced expression of H19 down-regulated the expression of β-Catenin and its downstream targets c-myc and Survivin in TMZ-treated glioma cells. Activation of Wnt/β-Catenin pathway by Licl treatment promoted EMT and enhanced chemoresistance to TMZ compared with TMZ+H19 shRNA group.

Conclusion: Taken together, our data suggest that H19 decreased chemoresistance of glioma cells to TMZ by suppressing EMT via the inhibition of Wnt/β-Catenin pathway. Our study might represent a novel therapeutic target for TMZ-resistant glioma.

Keywords: EMT; LncRNA-H19; Wnt/β-Catenin; chemoresistance; glioma; temozolomide.

Figure 1

H19 is up-regulated in TMZ-resistant glioma cells and silencing of H19 decreases chemoresistance of glioma cells to TMZ. Notes: (A) Relative expression of H19 was detected in glioma cells (U-251, M059J) and TMZ-resistant glioma cells (U-251TMZ, M059JTMZ) by qRT-PCR. ***P<0.001 compared with U-251 or M059J, respectively. (B) Relative expression of H19 was detected in U-251TMZ or M059JTMZ cells transfected with H19 scramble or H19 shRNA and in the control group, by qRT-PCR. *P<0.05 compared with H19 scramble group. (C, D) Cell viability was measured by MTT assay. (E–H) Cell apoptosis rate was measured by flow cytometric analysis. The bars show mean ± SD of three independent experiments. *P<0.05 compared with control group, ^#P<0.05 compared with TMZ+H19 scramble group. Abbreviations: DMSO, dimethyl sulfoxide; qRT-PCR, quantitative real-time polymerase chain reaction; TMZ, temozolomide.

Figure 2

Silencing of H19 inhibits EMT in glioma cells. Notes: U-251TMZ cells transfected with or without H19 scramble or H19 shRNA received TMZ treatment. (A–D) Relative protein level of E-cadherin, Vimentin, and ZEB1 was measured by Western blot with GAPDH as an endogenous reference. The bars show mean ± SD of three independent experiments. *P<0.05 compared with control group, ^#P<0.05 compared with TMZ+H19 scramble group. Abbreviations: EMT, epithelial-mesenchymal transition; TMZ, temozolomide.

Figure 3

Inducing EMT counteracts the inhibitory effect of H19 shRNA on chemoresistance of glioma cells to TMZ. Notes: U-251TMZ cells transfected with H19 shRNA were treated with TMZ combined with or without TGF-β1. (A, B) Relative protein level of E-cadherin, Vimentin, and ZEB1 was measured by Western blot with GAPDH as an endogenous reference. (C) Cell viability was measured by MTT assay. (D) Cell apoptosis rate was measured by flow cytometric analysis. The bars show mean ± SD of three independent experiments. *P<0.05 compared with control group, ^#P<0.05, ^##P<0.01 compared with TMZ-treated group, ^$P<0.05, ^$$P<0.01 compared with TMZ+H19 shRNA group. Abbreviations: DMSO, dimethyl sulfoxide; EMT, epithelial-mesenchymal transition; TMZ, temozolomide.

Figure 4

Silencing of H19 decreases chemoresistance through suppressing EMT via the Wnt/β-Catenin pathway. Notes: U-251TMZ cells transfected with H19 shRNA were treated with TMZ combined with or without Licl. (A, B) Relative protein level of β-Catenin, c-myc, and Survivin was measured by Western blot with GAPDH as an endogenous reference. (C–F) Relative protein level of β-Catenin, E-cadherin, and Vimentin was measured by Western blot with GAPDH as an endogenous reference. (G) Cell viability was measured by MTT assay. (H) Cell apoptosis rate was measured by flow cytometric analysis. The bars show mean ± SD of three independent experiments. *P<0.05 compared with control group, ^#P<0.05 compared with TMZ-treated group, ^$P<0.05, ^$$P<0.01 compared with TMZ+H19 shRNA group. Abbreviations: DMSO, dimethyl sulfoxide; EMT, epithelial-mesenchymal transition; TMZ, temozolomide.