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An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells
Published on: May 21, 2019
Improving Gemcitabine Sensitivity in Pancreatic Cancer Cells by Restoring miRNA-217 Levels
Concetta Panebianco1, Nadia Trivieri2, Annacandida Villani1
1Gastroenterology Unit, Fondazione IRCCS Casa Sollievo della Sofferenza, 71013 San Giovanni Rotondo, Italy.
Abstract:
Chemoresistance is a major problem in the therapeutic management of pancreatic cancer, concurring to poor clinical outcome. A number of mechanisms have been proposed to explain resistance to gemcitabine, a standard of care for this malignancy, among which is included aberrant miRNA expression. In the current study, we investigated the role of miR-217, which is strongly down-regulated in cancerous, compared to normal, pancreatic tissues or cells, in sensitizing human pancreatic cancer cell lines to this drug. The low expression of miR-217 in pancreatic cancer patients was confirmed in two gene expression datasets (GSE41372 and GSE60980), and the prognostic value of two target genes (ANLN and TRPS1), was estimated on clinical data from the Tumor Cancer Genome Atlas (TCGA). Transfecting miR-217 mimic in pancreatic cancer cells reduced viability, enhanced apoptosis, and affected cell cycle by promoting a S phase arrest in gemcitabine-treated cells. Moreover, in drug-exposed cells subjected to miR-217 forced expression, a down-regulation for several genes involved in cancer drug resistance was observed, many of which are cell cycle regulators, such as CCND1, CCNE1, CDK2, CDKN1A, CDKN1B, while others, such as ARNT, BRCA1, BRCA2, ELK1, EGFR, ERBB4, and RARA are involved in proliferation and cell cycle progression. Our results support the notion that miR-217 enhances pancreatic cancer sensitivity to gemcitabine, mainly impairing cell cycle progression.
Insights
MicroRNA-217 (miR-217) down-regulation contributes to gemcitabine resistance in pancreatic cancer. Restoring miR-217 enhances drug sensitivity by impairing cell cycle progression and affecting key cancer-related genes.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Chemoresistance to gemcitabine significantly impacts pancreatic cancer patient outcomes.
- Aberrant microRNA (miRNA) expression is a proposed mechanism underlying gemcitabine resistance.
- miR-217 is notably downregulated in pancreatic cancer tissues and cells.
Purpose of the Study:
- To investigate the role of miR-217 in sensitizing human pancreatic cancer cell lines to gemcitabine.
- To confirm the low expression of miR-217 in pancreatic cancer patients.
- To evaluate the prognostic value of miR-217 target genes ANLN and TRPS1.
Main Methods:
- Analysis of gene expression datasets (GSE41372, GSE60980) to confirm miR-217 downregulation.
- Prognostic value estimation of ANLN and TRPS1 using TCGA clinical data.
- Transfection of miR-217 mimic in pancreatic cancer cells followed by gemcitabine treatment.
- Assessment of cell viability, apoptosis, and cell cycle.
- Gene expression analysis of drug resistance-related genes.
Main Results:
- Forced expression of miR-217 reduced pancreatic cancer cell viability and enhanced apoptosis.
- miR-217 induced S phase arrest in gemcitabine-treated cells.
- Downregulation of multiple genes involved in drug resistance, proliferation, and cell cycle regulation (e.g., CCND1, CCNE1, CDK2, CDKN1A, CDKN1B, ARNT, BRCA1, BRCA2, ELK1, EGFR, ERBB4, RARA) was observed upon miR-217 restoration.
Conclusions:
- miR-217 plays a crucial role in enhancing pancreatic cancer sensitivity to gemcitabine.
- The sensitizing effect of miR-217 is primarily mediated by the impairment of cell cycle progression.
- Restoring miR-217 levels represents a potential therapeutic strategy for overcoming gemcitabine resistance in pancreatic cancer.
