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Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma
Published on: March 13, 2013
Aberrant microRNAs expression in CD133⁺/CD326⁺ human lung adenocarcinoma initiating cells from A549
Sheng Lin1, Jian-Guo Sun, Jing-Bo Wu
1Institute of Cancer, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China.
Abstract:
Increasing evidence demonstrates that miRNAs are involved in the dysregulation of tumor initiating cells (TICs) in various tumors. Due to a lack of definitive markers, cell sorting is not an ideal separation method for lung adenocarcinoma initiating cells. In this study, we combined paclitaxel with serum-free medium cultivation (inverse-induction) to enrich TICs from A549 cells, marked by CD133/CD326, defined features of stemness. We next investigated aberrant microRNAs in this subpopulation compared to normal cells with miRNA microarray and found that 50 miRNAs exhibited a greater than 2-fold change in expression. As further validation, 10 miRNAs were chosen to perform quantitative RT-PCR on the A549 cell line and primary samples. The results suggest that aberrant expression of miRNAs such as miR-29ab, miR-183, miR-17-5p and miR-127-3P may play an important role in regulating the bio-behavior of TICs.
Insights
MicroRNAs (miRNAs) are implicated in tumor-initiating cells (TICs). This study enriched lung adenocarcinoma TICs using paclitaxel and serum-free medium, identifying 50 aberrant miRNAs potentially regulating TIC behavior.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- MicroRNAs (miRNAs) are increasingly recognized for their role in the dysregulation of tumor-initiating cells (TICs) across various cancers.
- Lung adenocarcinoma TICs pose a challenge for isolation due to the lack of definitive markers, hindering effective therapeutic strategies.
Purpose of the Study:
- To enrich and characterize tumor-initiating cells (TICs) from lung adenocarcinoma.
- To identify aberrant microRNAs (miRNAs) associated with enriched TICs and investigate their potential role in regulating TIC bio-behavior.
Main Methods:
- Enrichment of TICs from A549 lung adenocarcinoma cells using a combination of paclitaxel and serum-free medium (inverse-induction).
- Identification of TICs by CD133 and CD326 markers, indicative of stemness features.
- miRNA microarray analysis to detect differentially expressed miRNAs between enriched TICs and normal cells.
- Quantitative RT-PCR validation of selected miRNAs in cell lines and primary samples.
Main Results:
- Enrichment of lung adenocarcinoma TICs was achieved using the inverse-induction method.
- miRNA microarray analysis revealed over 50 miRNAs with a greater than 2-fold change in expression in TICs compared to normal cells.
- Quantitative RT-PCR confirmed the aberrant expression of specific miRNAs, including miR-29ab, miR-183, miR-17-5p, and miR-127-3p.
Conclusions:
- Aberrantly expressed miRNAs, such as miR-29ab, miR-183, miR-17-5p, and miR-127-3p, are associated with lung adenocarcinoma tumor-initiating cells.
- These miRNAs may play a significant role in regulating the biological behavior and stemness properties of TICs.
- The findings provide insights into the molecular mechanisms underlying TIC regulation and suggest potential therapeutic targets.
