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Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells
Published on: May 9, 2020
Knockdown of calponin 2 suppressed cell growth in gastric cancer cells
Jianwei Hu1, Wenjuan Xie2, Lingyue Shang2
11 Endoscopy Center and Department of General Surgery, Zhongshan Hospital of Fudan University, Shanghai, P.R. China.
Abstract:
Calponin family members are actin filament-associated regulatory proteins with distinct expression patterns. Previous studies on CNN2 (calponin 2) have demonstrated that CNN2 is expressed in a broad range of tissues and cell types, exhibiting potential regulatory roles in a number of cellular activities, including cell proliferation, cell migration, and platelet adhesion. In this work, we found that both messenger RNA and protein expression levels of CNN2 were remarkably upregulated in 60%-70% of gastric cancer tissues by comparison with those of neighboring non-tumorous mucosa. By utilizing specific shCNN2 (small hairpin RNA targeting CNN2), the potential role of CNN2 in regulating AGS gastric cancer cell growth was then further investigated. AGS cells infected with shCNN2 exhibited significantly decreased cell growth ability by comparison with control cells in vitro. Moreover, while there was no obvious difference in cell cycle distribution between two groups, enhanced cell apoptosis was detected in cells with reduced CNN2 expression. Consistently, caspase 3/7 activity was also remarkably activated upon shCNN2 lentivirus infection. Taken together, our results demonstrated that knockdown of endogenous CNN2 in AGS cells could significantly activate cell apoptosis pathway and therefore suppress cell growth in vitro. The deletion of CNN2 might be a potential therapeutic approach to inhibit aggressive growth of gastric cancer.
Insights
Calponin 2 (CNN2) is upregulated in gastric cancer. Reducing CNN2 levels in gastric cancer cells significantly inhibits their growth by promoting apoptosis, suggesting CNN2 as a potential therapeutic target.
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- Calponin family members are actin-associated regulatory proteins.
- Calponin 2 (CNN2) has known roles in cell proliferation, migration, and platelet adhesion.
- CNN2 expression patterns are distinct across various tissues and cell types.
Purpose of the Study:
- To investigate the role of CNN2 in gastric cancer.
- To determine the effect of CNN2 knockdown on gastric cancer cell growth and apoptosis.
Main Methods:
- Quantitative analysis of CNN2 mRNA and protein expression in gastric cancer tissues.
- Utilized small hairpin RNA (shCNN2) to reduce CNN2 expression in AGS gastric cancer cells.
- Assessed cell growth, cell cycle distribution, apoptosis, and caspase 3/7 activity.
Main Results:
- CNN2 mRNA and protein were upregulated in 60%-70% of gastric cancer tissues compared to non-tumorous mucosa.
- Knockdown of CNN2 in AGS cells significantly reduced cell growth in vitro.
- Reduced CNN2 expression led to enhanced apoptosis and activated caspase 3/7 activity.
Conclusions:
- Endogenous CNN2 knockdown suppresses gastric cancer cell growth by activating apoptosis.
- CNN2 upregulation is implicated in aggressive gastric cancer growth.
- Deletion or inhibition of CNN2 may represent a potential therapeutic strategy for gastric cancer.
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