ABSTRACT
BACKGROUND: The use of high-throughput sequencing data has improved the results of genomic analysis due to the resolution of mapping algorithms. Although several tools for copy-number variation calling in whole genome sequencing have been published, the noisy nature of sequencing data is still a limitation for accuracy and concordance among such tools. To assess the performance of PennCNV original algorithm for array data in whole genome sequencing data, we processed mapping (BAM) files to extract coverage, representing log R ratio (LRR) of signal intensity, and B allele frequency (BAF). RESULTS: We used high quality sample NA12878 from the recently reported NIST database and created 10 artificial samples with several CNVs spread along all chromosomes. We compared PennCNV-Seq with other tools with general deletions and duplications, as well as for different number of copies and copy-neutral loss-of-heterozygosity (LOH). CONCLUSION: PennCNV-Seq was able to find correct CNVs and can be integrated in existing CNV calling pipelines to report accurately the number of copies in specific genomic regions.
Subject(s)
Algorithms , DNA Copy Number Variations/genetics , Genome, Human , Whole Genome Sequencing , Databases, Nucleic Acid , Gene Deletion , Gene Duplication , Gene Frequency/genetics , Genome-Wide Association Study , Humans , Loss of Heterozygosity/genetics , Markov Chains , Polymorphism, Single Nucleotide/genetics , Reproducibility of ResultsABSTRACT
Aberrations in the apoptotic mechanisms that cause excessive or deficient programmed cell death have been linked to a wide array of pathological conditions. In this study, using real-time reverse transcriptase-PCR, we analyzed the expression of apoptosis-regulating miRNAs (miR-15a, miR-16, miR-17-5p, miR-20a, miR-21, miR-29a, and miR-34a) in 20 oral squamous cell carcinoma and 5 normal oral mucosa tissue samples. Bioinformatic algorithms were used to identify the target genes of these miRNAs (BCL2, CASP2, CASP7, CASP8, DIABLO). The expression transcript levels of the target genes were measured in 50 oral squamous cell carcinoma and 10 normal oral mucosa tissue samples. We observed downregulation of miR-15a, miR-29a, and miR-34a in 50, 75, and 70% of samples, respectively. miR-16, miR-17-5p, miR-20a, and miR-21 expression was normal in 80, 75, 90, and 60% of samples, respectively. BCL2 transcripts were downregulated in 60% of samples, and normal-like expression was observed for CASP2, CASP7, CASP8, and DIABLO transcripts in 66, 82, 68, and 60% of samples, respectively. BCL2 expression was negative to weak, and that of proteins CASP2, CASP7, CASP8, and DIABLO was moderate to strong. Our study provides evidence of alterations in the expression of apoptosis-regulating miRNAs and genes in the apoptotic pathway, demonstrating that regulation of apoptosis is a hallmark of oral squamous cell carcinoma pathogenesis.
Subject(s)
Apoptosis/genetics , Carcinoma, Squamous Cell/genetics , Gene Expression Regulation, Neoplastic , MicroRNAs/genetics , Mouth Neoplasms/genetics , Apoptosis Regulatory Proteins , Carcinoma, Squamous Cell/metabolism , Caspase 2/genetics , Caspase 2/metabolism , Caspase 7/genetics , Caspase 7/metabolism , Caspase 8/genetics , Caspase 8/metabolism , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Female , Humans , Immunohistochemistry , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Male , Middle Aged , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Mouth Neoplasms/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
HER-2-positive breast cancers frequently sustain elevated AKT/mTOR signaling, which has been associated with resistance to doxorubicin treatment. Here, we investigated whether rapamycin, an mTOR inhibitor, increased the sensitivity to doxorubicin therapy in two HER-2-overexpressing cell lines: C5.2, which was derived from the parental HB4a by transfection with HER-2 and SKBR3, which exhibits HER-2 amplification. The epithelial mammary cell line HB4a was also analyzed. The combined treatment using 20 nmol/L of rapamycin and 30 nmol/L of doxorubicin arrested HB4a and C5.2 cells in S to G(2)-M, whereas SKBR3 cells showed an increase in the G(0)-G(1) phase. Rapamycin increased the sensitivity to doxorubicin in HER-2-overexpressing cells by approximately 2-fold, suggesting that the combination displayed a more effective antiproliferative action. Gene expression profiling showed that these results might reflect alterations in genes involved in canonical pathways related to purine metabolism, oxidative phosphorylation, protein ubiquitination, and mitochondrial dysfunction. A set of 122 genes modulated by the combined treatment and specifically related to HER-2 overexpression was determined by finding genes commonly regulated in both C5.2 and SKBR3 that were not affected in HB4a cells. Network analysis of this particular set showed a smaller subgroup of genes in which coexpression pattern in HB4a cells was disrupted in C5.2 and SKBR3. Altogether, our data showed a subset of genes that might be more robust than individual markers in predicting the response of HER-2-overexpressing breast cancers to doxorubicin and rapamycin combination.
