Silencing of IL13RA2 promotes partial epithelial-mesenchymal transition in hepatocellular carcinoma via ERK signaling pathway activation

Abstract

Lack of insight into the mechanisms underlying hepatocellular carcinoma (HCC) metastasis has hindered the development of curative treatments. Overexpression of interleukin-13 receptor alpha 2 (IL13RA2) has been reported to contribute to invasion and metastasis in several tumors. However, the role of IL13RA2 in HCC remains to be characterized. In this study, we identified that low expression of IL13RA2 is associated with poor survival of patients with HCC, and demonstrated that IL13RA2 knockdown endows HCC cells with invasive potential. Mechanistically, silencing of IL13RA2 promotes partial epithelial-mesenchymal transition via increasing extracellular signal-regulated kinase phosphorylation in HCC. Collectively, our results suggest that IL13RA2 may have potential as a prognostic biomarker for HCC.

Keywords: ERK signaling; epithelial-mesenchymal transition; hepatocellular carcinoma; interleukin-13 receptor alpha 2.

Figure 1

Low expression of IL13RA2 correlates with poor prognosis in patients with HCC. Kaplan–Meier plot of the correlation between IL13RA2 expression and OS, using TCGA, GEO: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE20017 and GEO: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE9843 integrated databases. The OS of the IL13RA2 high‐expression group was better than that of the IL13RA2 low‐expression group (P = 0.0051).

Figure 2

The expression of IL13RA2 in HCC. (A) TCGA analysis of IL13RA2 expression in human HCC and normal hepatic tissue. IL13RA2 expressed higher in normal hepatic tissue. (B) Quantitative RT‐PCR analysis of IL13RA2 mRNA expression in HCC cell lines and normal hepatocyte cell line (L02). The IL13RA2 mRNA expressed higher in MHCC97L cells, MHCC97H cells and HCCLM3 cells. (C) Western blot analysis of IL13RA2 protein expression in HCC cell lines and normal hepatocyte cell line (L02). The IL13RA2 expressed higher in MHCC97H cells and HCCLM3 cells. *P < 0.05; ***P < 0.001 by Student’s t‐test. Error bars represent SD. n ≥ 3 independent experiments per condition.

Figure 3

IL13RA2 silencing enhances cell proliferation and migration, but inhibits cell apoptosis. (A) CCK‐8 assay for cell proliferation of MHCC97H‐shIL13RA2 cells and HCCLM3‐shIL13RA2 cells compared with their vector control. IL13RA2 knockdown increased cell proliferation of HCCLM3, but not of MHCC97H. (B) Flow cytometry analysis of apoptosis of MHCC97H‐shIL13RA2 cells and HCCLM3‐shIL13RA2 cells compared with their vector control. IL13RA2 knockdown decreased the total and early apoptosis rates of MHCC97H and HCCLM3 cells. (C, D) Wound‐healing assays (original magnification ×40; scale bars represent 200 µm) (C) and Transwell migration assays (original magnification ×100; scale bars represent 100 µm) (D) for cell migration in the indicated group. IL13RA2 knockdown showed obvious promotion of migration abilities in MHCC97H and HCCLM3 cells. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, by Student’s t‐test. Error bars represent SD. n ≥ 3 independent experiments per condition. 7‐AAD, 7‐aminoactinomycin D.

Figure 4

IL13RA2 knockdown induces EMT via ERK phosphorylation. (A) E‐cadherin, N‐cadherin and Vimentin were detected by western blot in MHCC97H‐shIL13RA2 cells and HCCLM3‐shIL13RA2 cells compared with their vector control. E‐cadherin was significantly decreased in the IL13RA2 knockdown group of MHCC97H cells, with N‐cadherin and Vimentin increased, whereas in the IL13RA2 knockdown group of HCCLM3 cells, N‐cadherin and Vimentin were significantly up‐regulated, with no loss of E‐cadherin. (B) Western blot detection of Erk 1/2 and p‐Erk 1/2 protein level. IL13RA2 silencing increased the expression of p‐Erk 1/2 obviously in MHCC97H and HCCLM3 cells.