COX7A1 suppresses the viability of human non-small cell lung cancer cells via regulating autophagy

Abstract

COX7A1 is a subunit of cytochrome c oxidase, and plays an important role in the super-assembly that integrates peripherally into multi-unit heteromeric complexes in the mitochondrial respiratory chain. In recent years, some researchers have identified that COX7A1 is implicated in human cancer cell metabolism and therapy. In this study, we mainly explored the effect of COX7A1 on the cell viability of lung cancer cells. COX7A1 overexpression was induced by vector transfection in NCI-H838 cells. Cell proliferation, colony formation and cell apoptosis were evaluated in different groups. In addition, autophagy was analyzed by detecting the expression level of p62 and LC3, as well as the tandem mRFP-GFP-LC3 reporter assay respectively. Our results indicated that the overexpression of COX7A1 suppressed cell proliferation and colony formation ability, and promoted cell apoptosis in human non-small cell lung cancer cells. Besides, the overexpression of COX7A1 blocked autophagic flux and resulted in the accumulation of autophagosome via downregulation of PGC-1α and upregulation of NOX2. Further analysis showed that the effect of COX7A1 overexpression on cell viability was partly dependent of the inhibition of autophagy. Herein, we identified that COX7A1 holds a key position in regulating the development and progression of lung cancer by affecting autophagy. Although the crosstalk among COX7A1, PGC-1α and NOX2 needs further investigation, our study provides a novel insight into the therapeutic action of COX7A1 against human non-small cell lung cancer.

Keywords: COX7A1; NOX2; PGC-1α; autophagy; cell viability; non-small cell lung cancer.

Figure 1

The overexpression of COX7A1 suppressed cell viability and promoted cell apoptosis in human H838 cells. A, Western blot detection of COX7A1 overexpression induced by vector transfection. B, Evaluation of cell proliferation in the Control and COX7A1 overexpression group. C, Colony formation ability assay. D, Detection of apoptosis genes (Bax and Caspase 3) using western blot. E and F, Cell apoptosis assay using Annexin V‐FITC/PI staining (E) and Tunel staining (F). H838 cells were transfected with pCI‐COX7A1 to induce the overexpression of COX7A1, and an empty vector was used in the control group. The cells were harvested for detection after being transfected for 24 hours. Results are expressed as mean ± SD. A t test was used to compare the different groups, and P < .05 was considered statistically significant. *P < .05 compared with the Control group

Figure 2

COX7A1 induced the blocking of autophagy via downregulation of PGC‐1α. A, Effect of COX7A1 on PGC‐1, RIP140, and autophagy‐related proteins. B, Evaluation of PGC‐1α knockdown using western blot. C, Effect of PGC‐1α knockdown on the level of autophagy‐related proteins. D, Detection of apoptosis genes (Bax and Caspase 3) expression. E, Cell apoptosis assay using Tunel staining. Results are expressed as mean ± SD. A t test was used to compare the different groups, and P < .05 was considered statistically significant. *P < .05 compared with the Control group (group I)

Figure 3

Evaluation of autophagic flux using tandem mRFP‐GFP‐LC3 reporter. A, The fluorescence image of H838 cells after different transfection. Scale bar = 10 μm. B, Bar graphs represent the total number of mRFP‐GFP‐LC3 positive puncta and % of autolysosomes (red puncta) and autophagosome (yellow puncta) per cell. H838 cells were transfected with PGC‐1α siRNA first. After 24 hours, the cell were further transfected with pCI‐COX7A1 or ptf‐LC3 for another 24 hours. Then the cell samples in each group were applied for analysis. Results are expressed as mean ± SD. A t test was used to compare the different groups, and P < .05 was considered statistically significant. *P < .05 compared with the Control group (group I)

Figure 4

NOX2 knockdown abolishes the blockage of autophagy induced by COX7A1 overexpression. A, Evaluation of NOX2 knockdown using western blot. B, Effect of NOX2 knockdown on the level of autophagy‐related proteins. H838 cells were transfected with NOX2 siRNA first. After 24 hours, the cells were further transfected with pCI‐COX7A1 for another 24 hours to induce COX7A1 overexpression. Then the cell samples in each group were applied for analysis. Results are expressed as mean ± SD. A t test was used to compare the different groups, and P < .05 was considered statistically significant. *P < .05 compared with the Control group (group I)

Figure 5

The influence of COX7A1 on cell viability depends on the regulation of NOX2 partly. A, Evaluation of cell proliferation in different groups. B, Detection of apoptosis genes (Bax and Caspase 3) expression in each group. C, Cell apoptosis assay using Tunel staining. D, Colony formation ability assay. Results are expressed as mean ± SD. A t test was used to compare the different groups, and P < .05 was considered statistically significant. *P < .05 compared with Group I. #: P < .05 compared with Group II. &: P < .05 compared with Group III

Figure 6

Proposed model for the function of COX7A1 to suppressing the viability of human non‐small cell lung cancer cells via regulating autophagy. Autophagy could be inhibited by NOX2, and activated by PGC‐1α. Herein, the effect of COX7A1 on NOX2 and PGC‐1 is different. COX7A1 overexpression leads to the downregulation of PGC‐1α and upregulation of NOX2, which further results in the inhibition of autophagy totally