Array–comparative genomic hybridization (a-CGH) analysis
DNA copy number analysis was performed using an oligonucleotide
array-CGH platform (SurePrint G3 Human CGH Microarray 8x60K; Agilent
Technologies Inc., Santa Clara, CA, USA), using a previously established
protocol for FFPE samples 23, 24. DNA was isolated
using the standard phenol-chloroform method. Reference DNA was prepared
from the peripheral blood of a pool of ten healthy donors25. Equal amounts of tumor and reference genomic DNA
(1-2 µg) were digested and enzymatically labeled using the SureTag
Complete DNA Labeling Kit (Agilent Technologies, Inc., Santa Clara, CA,
USA) and hybridized to the arrays. The array data were analyzed with the
Feature Extraction (FE) v.10.10 software and Agilent CytoGenomics v.3.0
software (Agilent Technologies Inc., Santa Clara, CA, USA), using the
ADM-2 algorithm, threshold 6.0, and an aberration filter with a minimum
of > 3 probes 25. Copy number gains and
losses were defined as previously described 24.
Cytobands that showed copy number alterations (CNAs) in the
Ch11q-deleted locus in pre- and post-CT samples from three patients were
identified using Agilent CytoGenomics v.7.0 interval base reports
(Agilent Technologies, Inc., Santa Clara, CA, USA). Genes were extracted
from these cytobands using the UCSC genome browser 26and the RefSeq gene model. The online data mining software BioMart of
the Ensembl platform (http://m.ensembl.org/; release 101, August 2020)
was used to select the protein-coding genes within this region.
DIANA-miRPath v.3 and TarBase v.7.0 27, 28 were used
to identify the predicted miRNA targets. Only miRNA/mRNA targets that
had a TargetScan context score of -0.4 (default) were included.