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Updated: Dec 29, 2025
An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells
Published on: May 21, 2019
Identification of potential microRNAs and their targets in promoting gefitinib resistance by integrative network
Fushuang Zheng1, Hongyan Zhang1, Jibin Lu1
1Department of Thoracic Surgery, Shengjing Hospital of China Medical University, Shenyang 110004, China.
Background:
Non-small cell lung cancer (NSCLC) accounts for about 80-85% of lung cancers. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) such as gefitinib are considered the best choice for first-line treatment for the patients with NSCLC harboring EGFR-activating alterations. Nonetheless, 10-30% of patients may not obtain an objective response and may also experience rapid progression. The aim of our research, based on the integrative bioinformatics review, was to identify the possible miRNAs involved in gefitinib resistance.
Method:
A gefitinib-resistant network composed of 15 miRNAs and 34 targets were constructed by using the bioinformatics analyses of three microarray datasets. Of these miRNAs, effects of miR-342-3p on gefitinib resistance were investigated on a gefitinib-resistant cell model (A549/GR and PC/GR cells).
Results:
We reported that over-expression of miR-342-3p could significantly increase the resistance to gefitinib of A549/GR and PC9/GR cells and vice versa. Then, we recognized CPA4 as a target of hsa-miR-342-3p by a luciferase reporter assay. The increase in hsa-miR-342-3p levels led to a significant reduction in CPA4 protein expression. However, the opposite results were observed upon miR-342-3p knockdown. Finally, we found that enforced CPA4 expression partially reversed miR-342-3p effects in A549/GR cells.
Conclusions:
Collectively, these findings suggest that the upregulation of miR-342-3p contributes to gefitinib resistance by targeting CPA4, which may serve as a potential treatment option to overcome gefitinib resistance in patients with NSCLC.
Insights
Upregulation of miR-342-3p increases gefitinib resistance in non-small cell lung cancer (NSCLC) by targeting CPA4. This finding suggests miR-342-3p as a potential therapeutic target to overcome treatment resistance in NSCLC patients.
Area of Science:
- Oncology
- Molecular Biology
- Bioinformatics
Background:
- Non-small cell lung cancer (NSCLC) is a prevalent cancer type, with EGFR-TKIs like gefitinib being a primary treatment for EGFR-altered tumors.
- A significant subset of NSCLC patients exhibit resistance to gefitinib, necessitating research into underlying mechanisms.
- Identifying novel therapeutic targets is crucial for improving treatment outcomes in NSCLC.
Purpose of the Study:
- To investigate the role of microRNAs (miRNAs) in gefitinib resistance in NSCLC using bioinformatics analysis.
- To identify specific miRNAs and their targets involved in the development of gefitinib resistance.
Main Methods:
- Construction of a gefitinib-resistant network using bioinformatics analysis of three microarray datasets, identifying 15 miRNAs and 34 targets.
- Experimental validation of miR-342-3p's effect on gefitinib resistance in resistant NSCLC cell models (A549/GR and PC9/GR).
- Luciferase reporter assays to confirm CPA4 as a direct target of hsa-miR-342-3p.
Main Results:
- Overexpression of miR-342-3p significantly enhanced gefitinib resistance in A549/GR and PC9/GR cells, while knockdown decreased resistance.
- hsa-miR-342-3p was confirmed to target CPA4, with increased miR-342-3p levels reducing CPA4 protein expression.
- Enforced expression of CPA4 partially reversed the gefitinib resistance induced by miR-342-3p.
Conclusions:
- Upregulation of miR-342-3p contributes to gefitinib resistance in NSCLC by targeting and downregulating CPA4.
- miR-342-3p and its interaction with CPA4 represent a potential therapeutic strategy to overcome gefitinib resistance in NSCLC patients.
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