TMEM158 promotes the proliferation and migration of glioma cells via STAT3 signaling in glioblastomas

Abstract

Glioblastoma is the most common primary intracranial malignant tumor in adults and has high morbidity and high mortality. TMEM158 has been reported to promote the progression of solid tumors. However, its potential role in glioma is still unclear. Here, we found that TMEM158 expression in human glioma cells in the tumor core was significantly higher than that in noncancerous cells at the tumor edge using bioinformatics analysis. Cancer cells in patients with primary GBMs harbored significantly higher expression of TMEM158 than those in patients with WHO grade II or III gliomas. Interestingly, regardless of tumor grading, human glioma samples that were IDH1-wild-type (IDH1-WT) exhibited higher expression of TMEM158 than those with IDH1-mutant (IDH1-Mut). We also illustrated that TMEM158 mRNA expression was correlated with poor overall survival in glioma patients. Furthermore, we demonstrated that silencing TMEM158 inhibited the proliferation of glioma cells and that TMEM158 overexpression promoted the migration and invasion of glioma cells by stimulating the EMT process. We found that the underlying mechanism involves STAT3 activation mediating TMEM158-driven glioma progression. In vivo results further confirmed the inhibitory effect of the TMEM158 downregulation on glioma growth. Collectively, these findings further our understanding of the oncogenic function of TMEM158 in gliomas, which represents a potential therapeutic target, especially for GBMs.

Fig. 1. TMEM158 mRNA is highly expressed in IDH1-WT GBMs and is correlated with poor prognosis in glioma patients.

A Expression profile of TMEM158 in 31 kinds of cancers and their paired normal tissues from TCGA database. B–D Relationship between TMEM158 mRNA expression and WHO glioma grades and IDH1 mutation status of glioma samples in the TCGA and CGGA databases. E–G Expression of TMEM158 in IDH1-Mut-codel LGG, IDH1-Mut-noncodel LGG, IDH1-WT LGG, IDH1-Mut GBM, and IDH1-WT GBM. H–J TCGA and CGGA datasets were used for survival analysis of the two groups of glioma patients with higher TMEM158 expression and lower TMEM158 expression in glioma patients. K–L Kaplan–Meier survival curves were used to analyze the overall survival of GBM patients with higher expression of TMEM158 and lower expression of TMEM158 in the TCGA Agilent and U133A databases. (^nsp > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 2. TMEM158 is related to glioma grade and is preferentially expressed in the core of glioma tissues.

A TMEM158 protein expression was detected in a tissue microarray (TMA) (nontumor, n = 2; WHO grade II, n = 12; WHO grade II, n = 12; WHO grade IV, n = 28). B Total IHC staining score of TMA was related to WHO glioma grades, and nontumor samples exhibited the lowest protein expression of TMEM158. C The intratumoral and peritumoral expression patterns of TMEM158. D–G Representative image of TMEM158 expression on nontumor, WHO II, WHO III, and WHO IV tumor samples. H–K Partial enlarged image of (D–G). L RT-PCR results show the expression of TMEM158 mRNA in different grades of gliomas. M Western blotting was used to determine expression of TMEM158 in 8 different glioma cell lines. N Expression of TMEM158 in glioma cell lines in the CCLE database. (^nsp > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 3. TMEM158 overexpression promotes the proliferation, migration, and invasion of glioma cells.

A–B RT-PCR results showing the transfection efficiency of lentiviral overexpression (OE-TMEM158) and knockdown (shRNA-TMEM158-1, sh-1; shRNA-TMEM158-2, sh-2) and the expression of TMEM158 in U87MG, U251MG, and TJ905 glioma cell lines. C–D CCK-8 results showing the proliferation of U87MG, U251MG, and TJ905 glioma cells in response to upregulating and downregulating TMEM158 expression. E, G Colony-formation assay results showing the colony-formation efficiency of glioma cells after overexpression of TMEM158. F, H Colony-formation assay results showing the colony formation efficiency of glioma cells after silencing TMEM158 expression. I–L The migration ability in U87MG, U251MG, and TJ905 glioma cells in response to overexpression and knockdown of TMEM158 was detected by Transwell migration assay. K and L Show the statistical histogram of the quantification of migrated cells after upregulation and downregulation of TMEM158, respectively. M–P The representative images of Transwell invasion assay for glioma cell lines after gain- and loss-of-function TMEM158. The number of invaded cells is shown in (O and P). Data in (K, L, O, and P) are shown as mean ± SD. Scale bar = 100 μm. (^nsp > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 4. TMEM158 was positively correlated with EMT process and STAT3 signaling.

A Volcano plot showing the differentially expressed genes (DEGs) related to TMEM158. We identified a total of 776 upregulated genes and 1040 downregulated genes. B GSEA and HALLMARK gene set enrichment analysis were used to enrich the pathways related to TMEM158-associated genes. C GSEA showed that TMEM158-associated genes were enriched in the EMT process. (NES = 2.89, p < 0.0001). D TMEM158-associated genes were enriched in the STAT3 signaling pathway by GSEA. (NES = 2.20, p < 0.0001) E–F Western blotting results show changes in TMEM158 and EMT marker expression (E-Ca, N-Ca, vimentin, and Snail) in U87MG, U251MG, and TJ905 glioma cells after overexpression and knockdown of TMEM158. G–H TMEM158, STAT3, and p-STAT3 protein levels in U87MG, U251MG, and TJ905 glioma cells after overexpression and knockdown TMEM158 detected by western blotting.

Fig. 5. TMEM158 mediates the proliferation, migration, and invasion of glioma cells by activating STAT3 signaling.

A–C CCK-8 results showing the proliferation of sh-NC + pcDNA, sh-NC + STAT3 pcDNA, sh-TMEM158 + pcDNA, and sh-TMEM158 + STAT3 pcDNA in the four groups of glioma cells. D Colony-formation assay results showing the colony formation efficiency of glioma cells after silencing TMEM158 and upregulating STAT3. E–F Transwell assay results showing that overexpression of STAT3 rescued the reduced migration and invasion caused by TMEM158 knockdown. G Western blotting results showing the expression of TMEM158, STAT3, p-STAT3, E-Ca, N-Ca, vimentin, and Snail in the four groups of glioma cells. (^nsp > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 6. Silencing TMEM158 impairs the invasion of GBM cells in vivo and suppresses tumor growth.

A Schematic diagram of experimental grouping, implantation, and bioluminescence imaging of the orthotopic xenograft model. B–C Bioluminescence imaging of tumor growth in animals. Signal intensities were quantified on days 7, 14, 21, and 28 after implantation. n = 7 per group. (****p < 0.0001) D Kaplan–Meier survival curves indicating the percentage survival of mice. E Representative images of H & E staining of the mouse cerebrum showing the tumor border. Immunohistochemical (IHC) staining for TMEM158, p-STAT3, and Ki67 in the samples.