Loss of SH3GL2 promotes the migration and invasion behaviours of glioblastoma cells through activating the STAT3/MMP2 signalling

Abstract

SH3GL2 (Src homology 3 (SH3) domain GRB2-like 2) is mainly expressed in the central nervous system and regarded as a tumour suppressor in human glioma. However, the molecular mechanism of the SH3GL2 protein involved in malignant behaviours of human glioma has not been elucidated. In this study, we tried to investigate the role of SH3GL2 in glioma cell migration and invasion and explore its underlined molecular mechanism. Firstly, we discovered that the protein level of SH3GL2 was widely decreased in the human glioma patients, especially in high-grade glioma tissues. Then, we determined the role of SH3GL2 in migration and invasion of glioma cells upon SH3GL2 knocking down and overexpressing. It was showed that knocking down of SH3GL2 promoted the migration and invasion of glioma cells, whereas overexpression of SH3GL2 inhibited them. Further study on molecular mechanism disclosed that silencing of SH3GL2 obviously activated the STAT3 (signal transducer and activator of transcription 3) signalling thereby promoting the expression and secretion of MMP2. On the contrary, overexpression of SH3GL2 had opposite effect. Taken together, the above results suggest that SH3GL2 suppresses migration and invasion behaviours of glioma cells through negatively regulating STAT3/MMP2 signalling and that loss of SH3GL2 may intensify the STAT3/MMP2 signalling thereby contributing to the migration and invasion of glioma cells.

Keywords: MMP2; SH3GL2; STAT3; glioma.

Figure 1

Expression of SH3GL2 in human glioma patients and glioma cell lines. (A) Total proteins isolated from non‐tumorous brain tissue and glioma tissues were analysed by Western blotting for assessment of SH3GL2. (B) Statistical chart showed the expression level of SH3GL2 in non‐tumorous brain tissue and the different grades of glioma tissues. The ratios indicate the levels of SH3GL2 to β‐actin levels with respect to each sample. (C) Representative images of SH3GL2 from non‐tumorous brain tissues and human gliomas (grades II‐IV) determined by immunohistochemistry, scale bars: 20 μm. (D) Statistical chart showed the expression level of SH3GL2 in non‐tumorous brain tissue and the different grades of glioma tissues. (E) Expression of SH3GL2 in non‐tumorous cell line (293T) and glioma cell lines (U251, U87, A172, U118, C6). *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 2

Silencing of SH3GL2 in U251 cells. (A) The GFP photographs showed the stable cell line that silencing of SH3GL2 in U251 cells, scale bars: 200 μm. (B) The silencing efficiency of SH3GL2 #1‐3 was examined by Western blotting. (C) The silencing efficiency of SH3GL2 #1‐3 was verified by RT‐PCR. *P < 0.05.

Figure 3

Down‐regulation of SH3GL2 promotes glioma cell migration and invasion. (A) Wound healing assay with control and SH3GL2 #1‐3 expressing U251 cells, scale bars: 500 μm. (B) Statistical analysis showed that inhibition of SH3GL2 increased glioma cell migration compared with control. (C) Transwell migration assay with control and SH3GL2 #1‐3 expressing U251 cells, scale bars: 200 μm. (D) Statistical analysis showed that inhibition of SH3GL2 increased glioma cell migration compared with control. (E) Transwell invasion assay with control and SH3GL2 #1‐3 expressing U251 cells, scale bars: 200 μm. (F) Statistical analysis showed that inhibition of SH3GL2 increased glioma cell invasion compared with control. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Overexpressing of SH3GL2 in U87 cells. (A) The overexpressing efficiency of 3*FLAG‐SH3GL2 was examined by Western blotting. (B) The overexpressing efficiency of 3*FLAG‐SH3GL2 was examined by RT‐PCR. *P < 0.05.

Figure 5

Overexpression of SH3GL2 inhibits glioma cell migration and invasion. (A) Wound healing assay with 3*FLAG‐Vector or 3*FLAG‐SH3GL2 transfected U87 cells, scale bars: 500 μm. (B) Statistical analysis showed that overexpression of SH3GL2 inhibited glioma cell migration compared with the control. (C) Transwell migration assay with 3*FLAG‐Vector or 3*FLAG‐SH3GL2 transfected U87 cells, scale bars: 200 μm. (D) Statistical analysis showed that overexpression of SH3GL2 inhibited glioma cell migration compared with the control. (E) Transwell invasion assay with 3*FLAG‐Vector or 3*FLAG‐SH3GL2 transfected U87 cells, scale bars: 200 μm. (F) Statistical analysis showed that overexpression of SH3GL2 inhibited glioma cell invasion compared with the control. **P < 0.01, ***P < 0.001.

Figure 6

SH3GL2 negatively regulates STAT3/MMP2 signalling pathway. (A) Representative blots showed the levels of STAT3, p‐STAT3 and MMP2 in control or SH3GL2 silenced U251 cells. (B) Representative blots showed the levels of STAT3, p‐STAT3 and MMP2 in 3*FLAG‐Vector or 3*FLAG‐SH3GL2 overexpressed U87 cells. (C) RT‐PCR showed that silencing of SH3GL2 significantly up‐regulated the mRNA of MMP2. (D) RT‐PCR showed that overexpression of SH3GL2 significantly down‐regulated the mRNA of MMP2. (E) Gelatin zymogram assay showed that silencing of SH3GL2 increased the secretion of MMP2, while overexpressing of SH3GL2 decreased it. (F) Western blotting results showed that the expression of MMP2 and p‐STAT3 were gradually decreased along with the increase in 3*FLAG‐SH3GL2 amounts. (G) Western blotting results showed that silencing of SH3GL2 induced up‐regulation of p‐STAT3 and MMP2 could be significantly blocked by the STAT3 inhibitor, HO‐3867. (H) Western blotting results showed that SH3GL2‐STAT3/MMP2 signalling cascade also existed in PTEN intact C6 cells line. *P < 0.05 and **P < 0.01.