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Depletion of C3orf1/TIMMDC1 inhibits migration and proliferation in 95D lung carcinoma cells.

Wu H, Wang W, Xu H - Int J Mol Sci (2014)

Bottom Line: We demonstrated that C3orf1 depletion significantly suppressed 95D cell growth and migration.We confirmed C3orf1 localization in the inner mitochondrial membrane and showed that mitochondrial viability, membrane potential, and ATPase activity were remarkably reduced upon depletion of C3orf1.Concurrently, expression of the migration-promoting gene NUPR1 was markedly reduced, as confirmed by real-time PCR.

View Article: PubMed Central - PubMed

Affiliation: School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China. huiling@ujs.edu.cn.

ABSTRACT
In our previous study, we identified an association of high expression of c3orf1, also known as TIMMDC1 (translocase of inner mitochondrial membrane domain-containing protein 1), with metastatic characteristics in lung carcinoma cells. To investigate the preliminary function and mechanism of this mitochondrial protein, we depleted C3orf1 expression by introducing siRNA into 95D lung carcinoma cells. We demonstrated that C3orf1 depletion significantly suppressed 95D cell growth and migration. We confirmed C3orf1 localization in the inner mitochondrial membrane and showed that mitochondrial viability, membrane potential, and ATPase activity were remarkably reduced upon depletion of C3orf1. Microarray data indicated that genes involved in regulation of cell death, migration, and cell-cycle arrest were significantly altered after C3orf1 depletion for 48 h. The expression of genes involved in focal adhesion, ECM-receptor interaction, and p53-signaling pathways were notably altered. Furthermore, cell-cycle arrest genes such as CCNG2 and PTEN as well as genes involved in cell migration inhibition, such as TIMP3 and COL3A1, were upregulated after C3orf1 depletion in 95D cells. Concurrently, expression of the migration-promoting gene NUPR1 was markedly reduced, as confirmed by real-time PCR. We conclude that C3orf1 is critical for mitochondrial function, migration, and proliferation in 95D lung carcinoma cells. Depletion of C3orf1 inhibited cell migration and cell proliferation in association with upregulation of genes involved in cell-cycle arrest and cell migration inhibition. These results suggest that C3orf1 (TIMMDC1) may be a viable treatment target for lung carcinoma, and that further study of the role of this protein in lung carcinoma pathogenesis is justified.

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C3orf1 localizes to the inner mitochondrial membrane of 95D cells and C3orf1 knockdown affects mitochondria-related functions; (A) Representative images of immunostaining with anti-C3orf1 antibody (in green) and anti-Timm9 antibody (in red). The nucleus was labeled with Hoechst33342 dye (in blue). Scales bar = 20 µm; (B) Results of mitochondrial viability assays on 95D cells subjected to control or c3orf1 siRNA treatment for 2 days (2d) and 3 days (3d). Data are represented as mean ± SD. The data were normalized as described above, and the average mitochondrial viability of 95D cells treated with ctrl siRNA was set to 1; **p < 0.01, n = 3; (C) Results of mitochondrial potential assays on 95D cells subjected to control or c3orf1 siRNA treatment for 2 days (2d) and 3 days (3d). Data are represented as mean ± SD. The data were normalized, and the average mitochondrial potential of 95D cells treated with ctrl siRNA was set to 1; *p < 0.05, n = 3; (D) Results of mitochondrial number assays in 95D cells subjected to control or c3orf1 siRNA treatment for 2 days (2d) and 3 days (3d). Data are represented as mean ± SD. The data were normalized, and the average mitochondrial number of 95D cells treated with ctrl siRNA was set to 1; n = 3; ns = no significant change in mitochondrial numbers between cells treated with control siRNA or c3orf1 siRNA; and (E) Results of ATPase activity assays on 95D cells subjected to control or c3orf1 siRNA treatment for 2 days (2d) and 3 days (3d). Data are represented as mean ± SD. The data were normalized, and the average ATPase activity of 95D cells treated with ctrl siRNA was set to 1; *p < 0.05, **p < 0.01, n = 3.
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ijms-15-20555-f003: C3orf1 localizes to the inner mitochondrial membrane of 95D cells and C3orf1 knockdown affects mitochondria-related functions; (A) Representative images of immunostaining with anti-C3orf1 antibody (in green) and anti-Timm9 antibody (in red). The nucleus was labeled with Hoechst33342 dye (in blue). Scales bar = 20 µm; (B) Results of mitochondrial viability assays on 95D cells subjected to control or c3orf1 siRNA treatment for 2 days (2d) and 3 days (3d). Data are represented as mean ± SD. The data were normalized as described above, and the average mitochondrial viability of 95D cells treated with ctrl siRNA was set to 1; **p < 0.01, n = 3; (C) Results of mitochondrial potential assays on 95D cells subjected to control or c3orf1 siRNA treatment for 2 days (2d) and 3 days (3d). Data are represented as mean ± SD. The data were normalized, and the average mitochondrial potential of 95D cells treated with ctrl siRNA was set to 1; *p < 0.05, n = 3; (D) Results of mitochondrial number assays in 95D cells subjected to control or c3orf1 siRNA treatment for 2 days (2d) and 3 days (3d). Data are represented as mean ± SD. The data were normalized, and the average mitochondrial number of 95D cells treated with ctrl siRNA was set to 1; n = 3; ns = no significant change in mitochondrial numbers between cells treated with control siRNA or c3orf1 siRNA; and (E) Results of ATPase activity assays on 95D cells subjected to control or c3orf1 siRNA treatment for 2 days (2d) and 3 days (3d). Data are represented as mean ± SD. The data were normalized, and the average ATPase activity of 95D cells treated with ctrl siRNA was set to 1; *p < 0.05, **p < 0.01, n = 3.

Mentions: We used the online bioinformatic software MITOPROT to determine that C3orf1 has a probability of 0.9271 for being a mitochondrial membrane transport protein. Therefore, we also investigated the localization of C3orf1 protein in 95D cells using immunostaining with antibodies that bind C3orf1 and TIMM9, which is an inner mitochondrial membrane marker. TIMM9 co-localized with C3orf1 protein (Figure 3A). We then further investigated the effect of C3orf1 depletion on mitochondrial viability, number of mitochondria, mitochondrial membrane potential, and ATPase activity in 95D cells. As shown in Figure 3B,C, mitochondrial viability and the membrane potential were significantly decreased upon C3orf1 depletion by 23.4% and 18.4% at 2 day, and 28.3% and 27.8% at 3 day (p < 0.01 and 0.05), respectively. However, there was no significant change in the number of mitochondria in 95D cells upon c3orf1 siRNA treatment. In addition, mitochondrial ATPase activity was measured to investigate the effect of C3orf1 depletion on mitochondrial ATP generation in 95D cells. The result shown in Figure 3E demonstrates that the ATPase activity in C3orf1 depleted 95D cells was reduced by 10.1% at 2 day and 23.3% at 3 day compared to that in control cells (p < 0.05 and 0.01), respectively. Therefore, we concluded that C3orf1 protein knockdown significantly affected mitochondrial functions in 95D lung carcinoma cells.


Depletion of C3orf1/TIMMDC1 inhibits migration and proliferation in 95D lung carcinoma cells.

Wu H, Wang W, Xu H - Int J Mol Sci (2014)

C3orf1 localizes to the inner mitochondrial membrane of 95D cells and C3orf1 knockdown affects mitochondria-related functions; (A) Representative images of immunostaining with anti-C3orf1 antibody (in green) and anti-Timm9 antibody (in red). The nucleus was labeled with Hoechst33342 dye (in blue). Scales bar = 20 µm; (B) Results of mitochondrial viability assays on 95D cells subjected to control or c3orf1 siRNA treatment for 2 days (2d) and 3 days (3d). Data are represented as mean ± SD. The data were normalized as described above, and the average mitochondrial viability of 95D cells treated with ctrl siRNA was set to 1; **p < 0.01, n = 3; (C) Results of mitochondrial potential assays on 95D cells subjected to control or c3orf1 siRNA treatment for 2 days (2d) and 3 days (3d). Data are represented as mean ± SD. The data were normalized, and the average mitochondrial potential of 95D cells treated with ctrl siRNA was set to 1; *p < 0.05, n = 3; (D) Results of mitochondrial number assays in 95D cells subjected to control or c3orf1 siRNA treatment for 2 days (2d) and 3 days (3d). Data are represented as mean ± SD. The data were normalized, and the average mitochondrial number of 95D cells treated with ctrl siRNA was set to 1; n = 3; ns = no significant change in mitochondrial numbers between cells treated with control siRNA or c3orf1 siRNA; and (E) Results of ATPase activity assays on 95D cells subjected to control or c3orf1 siRNA treatment for 2 days (2d) and 3 days (3d). Data are represented as mean ± SD. The data were normalized, and the average ATPase activity of 95D cells treated with ctrl siRNA was set to 1; *p < 0.05, **p < 0.01, n = 3.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4264183&req=5

ijms-15-20555-f003: C3orf1 localizes to the inner mitochondrial membrane of 95D cells and C3orf1 knockdown affects mitochondria-related functions; (A) Representative images of immunostaining with anti-C3orf1 antibody (in green) and anti-Timm9 antibody (in red). The nucleus was labeled with Hoechst33342 dye (in blue). Scales bar = 20 µm; (B) Results of mitochondrial viability assays on 95D cells subjected to control or c3orf1 siRNA treatment for 2 days (2d) and 3 days (3d). Data are represented as mean ± SD. The data were normalized as described above, and the average mitochondrial viability of 95D cells treated with ctrl siRNA was set to 1; **p < 0.01, n = 3; (C) Results of mitochondrial potential assays on 95D cells subjected to control or c3orf1 siRNA treatment for 2 days (2d) and 3 days (3d). Data are represented as mean ± SD. The data were normalized, and the average mitochondrial potential of 95D cells treated with ctrl siRNA was set to 1; *p < 0.05, n = 3; (D) Results of mitochondrial number assays in 95D cells subjected to control or c3orf1 siRNA treatment for 2 days (2d) and 3 days (3d). Data are represented as mean ± SD. The data were normalized, and the average mitochondrial number of 95D cells treated with ctrl siRNA was set to 1; n = 3; ns = no significant change in mitochondrial numbers between cells treated with control siRNA or c3orf1 siRNA; and (E) Results of ATPase activity assays on 95D cells subjected to control or c3orf1 siRNA treatment for 2 days (2d) and 3 days (3d). Data are represented as mean ± SD. The data were normalized, and the average ATPase activity of 95D cells treated with ctrl siRNA was set to 1; *p < 0.05, **p < 0.01, n = 3.
Mentions: We used the online bioinformatic software MITOPROT to determine that C3orf1 has a probability of 0.9271 for being a mitochondrial membrane transport protein. Therefore, we also investigated the localization of C3orf1 protein in 95D cells using immunostaining with antibodies that bind C3orf1 and TIMM9, which is an inner mitochondrial membrane marker. TIMM9 co-localized with C3orf1 protein (Figure 3A). We then further investigated the effect of C3orf1 depletion on mitochondrial viability, number of mitochondria, mitochondrial membrane potential, and ATPase activity in 95D cells. As shown in Figure 3B,C, mitochondrial viability and the membrane potential were significantly decreased upon C3orf1 depletion by 23.4% and 18.4% at 2 day, and 28.3% and 27.8% at 3 day (p < 0.01 and 0.05), respectively. However, there was no significant change in the number of mitochondria in 95D cells upon c3orf1 siRNA treatment. In addition, mitochondrial ATPase activity was measured to investigate the effect of C3orf1 depletion on mitochondrial ATP generation in 95D cells. The result shown in Figure 3E demonstrates that the ATPase activity in C3orf1 depleted 95D cells was reduced by 10.1% at 2 day and 23.3% at 3 day compared to that in control cells (p < 0.05 and 0.01), respectively. Therefore, we concluded that C3orf1 protein knockdown significantly affected mitochondrial functions in 95D lung carcinoma cells.

Bottom Line: We demonstrated that C3orf1 depletion significantly suppressed 95D cell growth and migration.We confirmed C3orf1 localization in the inner mitochondrial membrane and showed that mitochondrial viability, membrane potential, and ATPase activity were remarkably reduced upon depletion of C3orf1.Concurrently, expression of the migration-promoting gene NUPR1 was markedly reduced, as confirmed by real-time PCR.

View Article: PubMed Central - PubMed

Affiliation: School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China. huiling@ujs.edu.cn.

ABSTRACT
In our previous study, we identified an association of high expression of c3orf1, also known as TIMMDC1 (translocase of inner mitochondrial membrane domain-containing protein 1), with metastatic characteristics in lung carcinoma cells. To investigate the preliminary function and mechanism of this mitochondrial protein, we depleted C3orf1 expression by introducing siRNA into 95D lung carcinoma cells. We demonstrated that C3orf1 depletion significantly suppressed 95D cell growth and migration. We confirmed C3orf1 localization in the inner mitochondrial membrane and showed that mitochondrial viability, membrane potential, and ATPase activity were remarkably reduced upon depletion of C3orf1. Microarray data indicated that genes involved in regulation of cell death, migration, and cell-cycle arrest were significantly altered after C3orf1 depletion for 48 h. The expression of genes involved in focal adhesion, ECM-receptor interaction, and p53-signaling pathways were notably altered. Furthermore, cell-cycle arrest genes such as CCNG2 and PTEN as well as genes involved in cell migration inhibition, such as TIMP3 and COL3A1, were upregulated after C3orf1 depletion in 95D cells. Concurrently, expression of the migration-promoting gene NUPR1 was markedly reduced, as confirmed by real-time PCR. We conclude that C3orf1 is critical for mitochondrial function, migration, and proliferation in 95D lung carcinoma cells. Depletion of C3orf1 inhibited cell migration and cell proliferation in association with upregulation of genes involved in cell-cycle arrest and cell migration inhibition. These results suggest that C3orf1 (TIMMDC1) may be a viable treatment target for lung carcinoma, and that further study of the role of this protein in lung carcinoma pathogenesis is justified.

Show MeSH
Related in: MedlinePlus