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Oncogene Expression Analysis with Alterations in pH in a Pancreatic Ductal Cell Line
Renuka Goudshelwar1, Sheethal Galande1, Karuna Rupula2
1Translational Research Centre, Asian Healthcare Foundation, AIG Hospitals.
Journal of Visualized Experiments : Jove
|April 28, 2025
Summary
The acidic environment in chronic pancreatitis activates proto-oncogenes, potentially initiating pancreatic ductal adenocarcinoma (PDAC) development. This study reveals how reduced pH influences gene expression, offering insights into PDAC
Area of Science:
- Oncology
- Molecular Biology
- Gastroenterology
Background:
- Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer with a low survival rate, often linked to a challenging tumor microenvironment.
- Patients with chronic pancreatitis have a significantly higher risk of developing PDAC, with ductal pH dropping from 8.0 to 6.0 due to inflammation and bicarbonate deficiency.
- The acidic microenvironment in chronic pancreatitis presents a critical factor influencing PDAC initiation and progression.
Purpose of the Study:
- To investigate the impact of an acidic environment, mimicking chronic pancreatitis, on oncogene expression in human pancreatic ductal epithelial cells.
- To identify specific oncogenes and their expression levels under varying acidic conditions using next-generation sequencing.
Main Methods:
- Human pancreatic ductal epithelial cells were cultured at different pH levels (6.0, 6.5, 7.0, and 7.2) for 6 hours.
- Total RNA was extracted, mRNA was enriched, and gene expression was analyzed using next-generation sequencing (RNA-seq).
- Differentially expressed genes (Fold Change < ± 2.0) were identified and analyzed, with a focus on oncogenes.
Main Results:
- Exposure to acidic conditions (pH 6.0, 6.5, 7.0) resulted in significant upregulation of numerous genes compared to neutral pH.
- Specifically, at pH 6.0, 90 genes were upregulated, and 20 were downregulated; at pH 6.5, 148 genes were upregulated, and 14 were downregulated; at pH 7.0, 109 genes were upregulated, and 23 were downregulated.
- Four oncogenes were upregulated at pH 6.0, seven at pH 6.5, and seven at pH 7.0. Notably, lymphocyte cell-specific protein-tyrosine kinase (LCK), FGR proto-oncogene, Src family tyrosine kinase (FGR), and ArfGAP With SH3 domain, ankyrin repeat, and PH domain 3 (ASAP3) were consistently upregulated across all tested acidic pH levels.
Conclusions:
- The acidic microenvironment characteristic of chronic pancreatitis can activate proto-oncogenes in pancreatic ductal cells.
- This activation of oncogenes by acidity may play a crucial role in the initiation of pancreatic ductal adenocarcinoma.
- Targeting the acidic tumor microenvironment could be a potential therapeutic strategy for PDAC prevention or treatment in high-risk patients.
