Potential targets of TMEM176A in the growth of glioblastoma cells

Abstract

Background: Human transmembrane protein 176A (TMEM176A) is upregulated in several tumors. Growing evidence has suggested the high clinical value of TMEM176A as a biomarker for early tumor diagnosis. However, less is known about the function of TMEM176A in glioblastomas (GBMs).

Methods: In this study, we systematically analyzed the effect of TMEM176A knockdown and overexpression in GBM cells (U87, T98G and A172) on cell proliferation, cell cycle and cell apoptosis.

Results: Our results indicated that TMEM176A acted as a tumor-promoting factor in GBM cells. Moreover, a specific ERK1/2 inhibitor, U0126, suppressed the function of TMEM176A in GBM cells. Therefore, we proposed that TMEM176A may be involved in a pathway including ERK1/2 in the regulation of the cell cycle. Moreover, we also found that TMEM176A affected the expression of Bcl2 and played a central role in apoptosis of GBM cells.

Conclusion: Taken together, our results not only elucidated the multiple functions of TMEM176A in GBM cells but also provided a deep insight into the potential targets of TMEM176A in the growth of GBM cells.

Keywords: ERK1/2; TMEM176A; cell apoptosis; cell cycle; glioblastomas.

Figure 1

TMEM176A was upregulated in GBM tissues and cell lines. Notes: (A) The mRNA and protein levels of TMEM176A and TMEM176B in GBM tissues *P<0.05 vs Normal ***P<0.001 vs Normal. (B) TMEM176A expression was significantly increased in GBM tissues (n=529) when compared with normal brain tissues (n=10) of patients from the TCGA GBM dataset (***P<0.001 vs Normal). (C) Survival analysis of patients from the TCGA GBM dataset. (D) The mRNA and protein levels of TMEM176A in different GBM cell lines. **P<0.01, ***P<0.001 vs T98G. Abbreviations: GBM, glioblastoma; TCGA, The Cancer Genome Atlas.

Figure 2

Knockdown and overexpression of TMEM176A in GBM cells. Notes: (A) The mRNA and protein levels of TMEM176A following a knockdown by siRNAs in T98G and U87 cells, ***P<0.001 vs BLANK. (B) The mRNA and protein levels of TMEM176A following overexpression by oeTMEM176A in A172 cells, ***P<0.001 vs BLANK. Abbreviation: GBM, glioblastoma.

Figure 3

Knockdown of TMEM176A suppressed GBM cells growth. Notes: (A) Cell proliferation was detected 0, 24, 48, and 72 hours after transfection with siNC, siTMEM176A1-1 and siTMEM176A1-2 in U87 and T98G cells *P<0.05 vs BLANK, **P<0.01 vs BLANK, ***P<0.001 vs BLANK. (B) Cell cycle profile of cells transfected with siNC, siTMEM176A1-1 and siTMEM176A1-2 in U87 and T98G cells **P<0.01 vs BLANK. (C) Cell apoptosis profile of cells transfected with siNC, siTMEM176A1-1, and siTMEM176A1-2 in U87 and T98G cells. (D) The protein levels of Cyclin D1, P21, cleaved-Caspase-3 and Bcl2 in various transfected cells. ***P<0.001 vs BLANK. Abbreviation: GBM, glioblastoma.

Figure 3

Knockdown of TMEM176A suppressed GBM cells growth. Notes: (A) Cell proliferation was detected 0, 24, 48, and 72 hours after transfection with siNC, siTMEM176A1-1 and siTMEM176A1-2 in U87 and T98G cells *P<0.05 vs BLANK, **P<0.01 vs BLANK, ***P<0.001 vs BLANK. (B) Cell cycle profile of cells transfected with siNC, siTMEM176A1-1 and siTMEM176A1-2 in U87 and T98G cells **P<0.01 vs BLANK. (C) Cell apoptosis profile of cells transfected with siNC, siTMEM176A1-1, and siTMEM176A1-2 in U87 and T98G cells. (D) The protein levels of Cyclin D1, P21, cleaved-Caspase-3 and Bcl2 in various transfected cells. ***P<0.001 vs BLANK. Abbreviation: GBM, glioblastoma.

Figure 4

Knockdown of TMEM176A affects the signal transduction in GBM cells. Notes: (A) The protein level of signal transduction-related proteins in U87 cells transfected with siNC, siTMEM176A1-1, and siTMEM176A1-2, respectively, **P<0.01 vs BLANK, ***P<0.001 vs BLANK. (B) The protein level of signal transduction-related proteins in T98G cells transfected with siNC, siTMEM176A1-1 and siTMEM176A1-2, respectively, **P<0.01 vs BLANK, ***P<0.001 vs BLANK. Abbreviation: GBM, glioblastoma.

Figure 5

ERK1/2 inhibitor U0126 inhibits the function of TMEM176A. Notes: (A) Cell proliferation was detected 0, 24, 48, and 72 hours after transfection with oeNC, oeTMEM176A, oeNC+U0126 and oeTMEM176A+U0126 in A172 cells *P<0.05 vs oeNC, **P<0.01 vs oeNC, ***P<0.001 vs oeNC. (B) Cell cycle profile of cells transfected with oeNC, oeTMEM176A, oeNC+U0126 and oeTMEM176A+U0126 in A172 cells. **P<0.01 vs oeNC. (C) Cell apoptosis profile of cells transfected with oeNC, oeTMEM176A, oeNC+U0126 and oeTMEM176A+U0126 in A172 cells. **P<0.01 vs oeNC, ***P<0.001 vs oeNC. (D) The protein levels of Cyclin D1, P21, cleaved-Caspase-3, Bcl2, ERK1/2 and p-ERK1/2 in different transfected cells. ***P<0.001 vs oeNC. Abbreviation: GBM, glioblastoma.

Figure 6

Knockdown of TMEM176A in GBM cells inhibits tumor growth in vivo. Notes: T98G cells transfected with siRNA control (siNC) or siTMEM176A were subcutaneously injected into athymic nude mice. (A) The volume of the tumor was determined every 3 days for 33 days of each group. The growth of the tumor was clearly suppressed in nude mice injected with siTMEM176A cells. *P<0.05 vs siNC, **P<0.01 vs siNC, ***P<0.001 vs siNC. (B) Xenografts in nude mice at day 33 of each group (n=6). (C) Mice were sacrificed, and tumors were weighed at day 33. The six tumor tissues obtained from the siTMEM176A group were smaller and lighter than those of the siNC group, **P<0.01 vs siNC. (D) Protein levels of TMEM176A, Cyclin D1, P21, cleaved-Caspase-3, Bcl2, ERK1/2, p-ERK1/2 were analyzed by Western blot in xenografts from nude mice, ***P<0.001 vs siNC. Abbreviation: GBM, glioblastoma.