Knockdown of PAICS inhibits malignant proliferation of human breast cancer cell lines

Abstract

Background: Phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS), an enzyme required for de novo purine biosynthesis, is associated with and involved in tumorigenesis. This study aimed to evaluate the role of PAICS in human breast cancer, which remains the most frequently diagnosed cancer and the leading cause of cancer-related death among women in less developed countries.

Results: Lentivirus-based short hairpin RNA targeting PAICS specifically depleted its endogenous expression in ZR-75-30 and MDA-MB-231 breast cancer cells. Depletion of PAICS led to a significant decrease in cell viability and proliferation. To ascertain the mechanisms through which PAICS modulates cell proliferation, flow cytometry was performed, and it was confirmed that G1-S transition was blocked in ZR-75-30 cells through PAICS knockdown. This might have occurred partly through the suppression of Cyclin E and the upregulation of Cyclin D1, P21, and CDK4. Moreover, PAICS knockdown obviously promoted cell apoptosis in ZR-75-30 cells through the activation of PARP and caspase 3 and downregulation of Bcl-2 and Bcl-xl expression in ZR-75-30 cells.

Conclusions: These findings demonstrate that PAICS plays an essential role in breast cancer proliferation in vitro, which provides a new opportunity for discovering and identifying novel effective treatment strategies.

Keywords: Breast cancer; Cell apoptosis; Cell cycle; PAICS; Proliferation.

Fig. 1

Lentivirus-delivered shRNA targeting PAICS depletes endogenous expression in ZR-75-30 breast cancer cells. a Evaluation of the lentivirus transduction rate, which was more than 80% as estimated by fluorescence and light microscopy observations. b Quantitative analysis of PAICS knockdown efficiency in ZR-75-30 cells assessed by qRT-PCR. The β-actin gene was used as an internal control. And representative immunoblot showing PAICS knockdown efficiency determined. GAPDH was used as an internal control. Data are shown as mean ± SD (n = 3; t test). **p < 0.01; magnification, ×100

Fig. 2

Knockdown of PAICS inhibits viability and proliferation of ZR-75-30breast cancer cells. a MTT assays showing growth curves of ZR-75-30cells. The number of viable cells was substantially decreased in the siPAICS group compared to that in the siControl (siCon) and control (Con) group. b Representative colony formation showing clonogenic survival determined in ZR-75-30 cells. c The number of colonies was substantially decreased in the siPAICS group compared to that in the siCon group and the Con group. Data are shown as mean ± SD (n = 3; t test). ***p < 0.001; magnification, ×40

Fig. 3

Effect of PAICS endogenous depletion on MDA-MB-231. a The lentivirus transduction rate (more than 80%) as observed under fluorescence and light microscopy. b Quantitative qPCR and western blot showed the efficient knonkdown of PACIS in mRNA and protein level in ZR-75-30 cells. c MTT assays ompared growth curves of MDA-MB-231, before and after PAICS depletion. **p < 0.01; magnification, ×100

Fig. 4

Knockdown of PAICS arrests cell cycle progression in ZR-75-30 breast cancer cells. a Comparison of the cell population in G0/G1, S, and G2/M phases between siControl (siCon) and siPAICS groups, as assessed by flow cytometry. b The percentage of cells in G0/G1 phase was significantly higher in the siPAICS group than in the siCon group, whereas the proportion of cells in the S phase was simultaneously reduced. Data are shown as mean ± SD (n = 3; t test). ***p < 0.001

Fig. 5

Assessment of apoptosis after PAICS knockdown in ZR-75-30 breast cancer cells. Apoptosis was investigated by flow cytometric analysis. a Representative diagrams showing the analysis of apoptosis. b The percentages of AnnexinV-APC/7-AAD-positive apoptotic cells are presented. c Cell cycle-related and apoptosis-related molecules were detected by western blotting using specific antibodies after PAICS knockdown in ZR-75-30 cells. GAPDH was used as the loading control. Data are shown as mean ± SD (n = 3; t test). ***p < 0.001