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J Vis Exp. 2016 Dec 9;(118). doi: 10.3791/54714.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models.

Author information

1
Department of Pediatric Oncology/Hematology, VU University Medical Center; Department of Hematology, VU University Medical Center; Department of Medical Oncology, VU University Medical Center.
2
Department of Pediatric Oncology/Hematology, VU University Medical Center.
3
Department of Clinical Genetics, VU University Medical Center.
4
Department of Medical Oncology, VU University Medical Center.
5
Division of General and Transplant Surgery, Azienda Ospedaliera Universitaria Pisana, Universita' di Pisa.
6
Amsterdam Immunology and Rheumatology Center, VU University Medical Center.
7
Department of Pediatric Oncology/Hematology, VU University Medical Center; Princess Máxima Center for Pediatric Oncology.
8
Department of Medical Oncology, VU University Medical Center; Cancer Pharmacology Lab, AIRC Start-Up Unit, University of Pisa; Institute of Nanoscience and Nanotechnology, CNR-Nano; e.giovannetti@vumc.nl.

Abstract

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

PMID:
28060337
PMCID:
PMC5226372
[Available on 2018-12-09]
DOI:
10.3791/54714
[Indexed for MEDLINE]

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