J Breast Cancer.  2014 Sep;17(3):200-206. 10.4048/jbc.2014.17.3.200.

siRNA-Mediated Suppression of Synuclein gamma Inhibits MDA-MB-231 Cell Migration and Proliferation by Downregulating the Phosphorylation of AKT and ERK

• He J
• Xie N
• Yang J
• Guan H
• Chen W
• Wu H
• Yuan Z
• Wang K
• Li G
• Sun J
• Yu L

• KMID: 2286346
• DOI: http://doi.org/10.4048/jbc.2014.17.3.200

Abstract

PURPOSE
Synuclein-gamma (SNCG), which was initially identified as breast cancer specific gene 1, is highly expressed in advanced breast cancers, but not in normal or benign breast tissue. This study aimed to evaluate the effects of SNCG siRNA-treatment on breast cancer cells and elucidate the associated mechanisms.
METHODS
Vectors containing SNCG and negative control (NC) siRNAs were transfected into MDA-MB-231 cells; mRNA levels were determined by real-time polymerase chain reaction. Cell proliferation was evaluated using the MTT assay, cell migration was assessed by the Transwell assay, apoptosis and cell cycle analyses were conducted with the flow cytometer, and Western blot analysis was performed to determine the relative levels of AKT, ERK, p-AKT, and p-ERK expression.
RESULTS
SNCG mRNA levels were significantly reduced in MDA-MB-231 cells transfected with SNCG siRNA. Our results indicate that in SNCG siRNA-treated cells, cell migration and proliferation decreased significantly, apoptosis was induced, and the cell cycle was arrested. Western blot analysis indicated that the protein levels of p-AKT and p-ERK were much lower in the SNCG siRNA-treated groups, than in the control and NC groups.
CONCLUSION
SNCG siRNA could decrease the migration and proliferation of breast cancer cells by downregulating the phosphorylation of AKT and ERK.

Keyword

Breast neoplasms; Extracellular signal-regulated MAP kinases; Human SNCG protein; Proto-oncogene proteins c-akt; Small interfering RNA

MeSH Terms

Apoptosis
Blotting, Western
Breast
Breast Neoplasms
Cell Cycle
Cell Migration Assays
Cell Movement*
Cell Proliferation
Extracellular Signal-Regulated MAP Kinases
Phosphorylation*
Proto-Oncogene Proteins c-akt
Real-Time Polymerase Chain Reaction
RNA, Messenger
RNA, Small Interfering
Synucleins*
Extracellular Signal-Regulated MAP Kinases
Proto-Oncogene Proteins c-akt
RNA, Messenger
RNA, Small Interfering
Synucleins

Figure

• 

  Figure 1 The relative concentration of breast cancer specific gene 1 mRNA in each group. Synuclein-γ (SNCG) and β-actin mRNA levels were determined by fluorescent quantitative polymerase chain reaction. Levels of SNCG mRNA were found to be much lower in SNCG siRNA-1 and SNCG siRNA-2 groups than negative control (NC) (p<0.01, respectively) and control groups (p<0.01, respectively). *p<0.01 vs. control group; †p<0.01 vs. NC group.

• 

  Figure 2 The relative migration rate of each group. The relative migration rate of Synuclein-γ (SNCG) siRNA-1 and SNCG siRNA-2 groups decreased significantly (p<0.05, respectively). NC=negative control. *p<0.05 vs. NC group.

• 

  Figure 3 MTT assay at 0, 24, 48, and 72 hours after transfection. To determine the amount of cells alive, absorbance was measured on 490 nm wavelength. The cell proliferation decreased significantly in Synuclein-γ (SNCG) siRNA-1 and SNCG siRNA-2 groups (p<0.01, p<0.01) while there was no difference between negative control (NC) group and control group (p>0.05). OD=optical density. *p<0.01 vs. control group; †p<0.01 vs. NC group.

• 

  Figure 4 Apoptosis of MDA-MB-231 in each group. Synuclein-γ (SNCG) siRNA induced apoptosis in MDA-MB-231 cells. (A) Control-1 group; (B) control-2 group; (C) negative control (NC)-1 group; (D) NC-2 group; (E) SNCG siRNA-1 group; (F) SNCG siRNA-2 group. PI=propidium iodide.

• 

  Figure 5 Cell cycle of MDA-MB-231 in each group. The cell cycle of MDA-MB-231 cells was arrested significantly. (A) Control group; (B) negative control (NC) group; (C) Synuclein-γ (SNCG) siRNA-1 group; (D) SNCG siRNA-2 group; (E) the histogram for the cell percentage of each mitotic phase in each group. *p<0.05 vs. control group; †p<0.05 vs NC group.

• 

  Figure 6 Western blotting. The levels of p-AKT and p-ERK were less in Synuclein-γ (SNCG) siRNA-1 and SNCG siRNA-2 groups than negative control (NC) and control groups while there were not significant differences in the expression of AKT and ERK among the four groups. (A) Western blotting of AKT, p-AKT, ERK, p-ERK and their corresponding internal reference (β-Actin); (B) the histogram for the content of proteins in each group.

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