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1.
Endocr Relat Cancer ; 30(8)2023 08 01.
Article in English | MEDLINE | ID: mdl-37130273

ABSTRACT

Differentiated thyroid cancers (DTCs) are malignancies that demonstrate strong but largely uncharacterized heritability. Germline variants that influence the risk of DTCs localize in disrupted in renal carcinoma 3 (DIRC3), a poorly described long non-coding RNA gene. Here, we investigated the function of DIRC3 in DTCs. Using patient-matched thyroid tissue pairs and The Cancer Genome Atlas data, we established that DIRC3 is downregulated in DTCs, whereas high expression of DIRC3 in tumors may reduce the risk of cancer recurrence. DIRC3 transcripts were enriched in cell nuclei, where they upregulated insulin-like growth factor binding protein 5 (IGFBP5), a gene that modulates the cellular response to insulin-like growth factor 1 (IGF1). Silencing DIRC3 in thyroid cancer cell lines (MDA-T32 and MDA-T120) had a dichotomous phenotypic influence: augmented cell migration and invasiveness, reduced apoptosis, but abrogated the MTT reduction rate. Transcriptomic profiling and gene rescue experiments indicated the functional redundancy in the activities of DIRC3 and IGFBP5. Moreover, the reduced level of DIRC3 enhanced the susceptibility of thyroid cancer cells to IGF1 stimulation and promoted Akt signaling via downregulation of the IGFBP5 protein. In conclusion, DIRC3 expression alters the phenotype of thyroid cancer cells and regulates the activity of the IGFBP5/IGF1/Akt axis. Our findings suggest that an interplay between DIRC3 and IGF signaling may play a role in promoting thyroid carcinogenesis.


Subject(s)
RNA, Long Noncoding , Thyroid Neoplasms , Humans , RNA, Long Noncoding/genetics , Proto-Oncogene Proteins c-akt/metabolism , Cell Line, Tumor , Neoplasm Recurrence, Local , Insulin-Like Growth Factor I/metabolism , Thyroid Neoplasms/genetics
2.
Int J Mol Sci ; 20(18)2019 Sep 06.
Article in English | MEDLINE | ID: mdl-31500099

ABSTRACT

Circular RNAs (circRNAs) are a class of single-stranded covalently closed RNA rings. Biogenesis of circRNAs, which may occur co-transcriptionally and post-transcriptionally via a back-splicing mechanism, requires the presence of complementary and/or inverted repeat sequences in introns flanking back-spliced exons and is facilitated by RNA-binding proteins. CircRNAs are abundant across eukaryotes; however, their biological functions remain largely speculative. Recently, they have been emerging as new members of a gene regulatory network and contributing factors in various human diseases including cancer, neurological, muscular and cardiovascular disorders. In this review, we present an overview of the current knowledge about circRNAs biogenesis and their aberrant expression in various human disorders. In particular, we focus on the latest discovery of circRNAs global upregulation in myotonic dystrophy type 1 (DM1) skeletal muscles and the role these prospective biomarkers might have for prognosis and therapeutic response in DM1.


Subject(s)
Myotonic Dystrophy/genetics , RNA, Circular/genetics , Alternative Splicing , Animals , Biomarkers , Disease Susceptibility , Gene Expression Regulation , Humans , Myotonic Dystrophy/metabolism , Myotonic Dystrophy/pathology , RNA, Circular/metabolism , RNA-Binding Proteins/metabolism
3.
Front Genet ; 10: 649, 2019.
Article in English | MEDLINE | ID: mdl-31428124

ABSTRACT

Splicing aberrations induced as a consequence of the sequestration of muscleblind-like splicing factors on the dystrophia myotonica protein kinase transcript, which contains expanded CUG repeats, present a major pathomechanism of myotonic dystrophy type 1 (DM1). As muscleblind-like factors may also be important factors involved in the biogenesis of circular RNAs (circRNAs), we hypothesized that the level of circRNAs would be decreased in DM1. To test this hypothesis, we selected 20 well-validated circRNAs and analyzed their levels in several experimental systems (e.g., cell lines, DM muscle tissues, and a mouse model of DM1) using droplet digital PCR assays. We also explored the global level of circRNAs using two RNA-Seq datasets of DM1 muscle samples. Contrary to our original hypothesis, our results consistently showed a global increase in circRNA levels in DM1, and we identified numerous circRNAs that were increased in DM1. We also identified many genes (including muscle-specific genes) giving rise to numerous (>10) circRNAs. Thus, this study is the first to show an increase in global circRNA levels in DM1. We also provided preliminary results showing the association of circRNA level with muscle weakness and alternative splicing changes that are biomarkers of DM1 severity.

4.
Nucleic Acids Res ; 46(17): e101, 2018 09 28.
Article in English | MEDLINE | ID: mdl-29878242

ABSTRACT

Genome editing technology based on engineered nucleases has been increasingly applied for targeted modification of genes in a variety of cell types and organisms. However, the methods currently used for evaluating the editing efficiency still suffer from many limitations, including preferential detection of some mutation types, sensitivity to polymorphisms that hamper mismatch detection, lack of multiplex capability, or sensitivity to assay conditions. Here, we describe qEva-CRISPR, a new quantitative method that overcomes these limitations and allows simultaneous (multiplex) analysis of CRISPR/Cas9-induced modifications in a target and the corresponding off-targets or in several different targets. We demonstrate all of the advantages of the qEva-CRISPR method using a number of sgRNAs targeting the TP53, VEGFA, CCR5, EMX1 and HTT genes in different cell lines and under different experimental conditions. Unlike other methods, qEva-CRISPR detects all types of mutations, including point mutations and large deletions, and its sensitivity does not depend on the mutation type. Moreover, this approach allows for successful analysis of targets located in 'difficult' genomic regions. In conclusion, qEva-CRISPR may become a method of choice for unbiased sgRNA screening to evaluate experimental conditions that affect genome editing or to distinguish homology-directed repair from non-homologous end joining.


Subject(s)
CRISPR-Cas Systems/physiology , DNA End-Joining Repair/genetics , Gene Editing/methods , Mutagenesis, Site-Directed/methods , Recombinational DNA Repair/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Evaluation Studies as Topic , Gene Editing/standards , HCT116 Cells , HEK293 Cells , HeLa Cells , Humans , K562 Cells , Mutagenesis, Site-Directed/standards , RNA, Guide, Kinetoplastida/genetics , Sequence Homology
5.
Sci Rep ; 8(1): 5885, 2018 04 12.
Article in English | MEDLINE | ID: mdl-29651162

ABSTRACT

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are human neuromuscular disorders associated with mutations of simple repetitive sequences in affected genes. The abnormal expansion of CTG repeats in the 3'-UTR of the DMPK gene elicits DM1, whereas elongated CCTG repeats in intron 1 of ZNF9/CNBP triggers DM2. Pathogenesis of both disorders is manifested by nuclear retention of expanded repeat-containing RNAs and aberrant alternative splicing. The precise determination of absolute numbers of mutant RNA molecules is important for a better understanding of disease complexity and for accurate evaluation of the efficacy of therapeutic drugs. We present two quantitative methods, Multiplex Ligation-Dependent Probe Amplification and droplet digital PCR, for studying the mutant DMPK transcript (DMPKexpRNA) and the aberrant alternative splicing in DM1 and DM2 human tissues and cells. We demonstrate that in DM1, the DMPKexpRNA is detected in higher copy number than its normal counterpart. Moreover, the absolute number of the mutant transcript indicates its low abundance with only a few copies per cell in DM1 fibroblasts. Most importantly, in conjunction with fluorescence in-situ hybridization experiments, our results suggest that in DM1 fibroblasts, the vast majority of nuclear RNA foci consist of a few molecules of DMPKexpRNA.


Subject(s)
Fibroblasts/metabolism , Multiplex Polymerase Chain Reaction/methods , Myotonic Dystrophy/genetics , Myotonin-Protein Kinase/genetics , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , 3' Untranslated Regions , Alternative Splicing , Exons , Fibroblasts/pathology , Gene Expression , Gene Expression Profiling , Humans , Introns , Multiplex Polymerase Chain Reaction/standards , Myotonic Dystrophy/classification , Myotonic Dystrophy/metabolism , Myotonic Dystrophy/pathology , Myotonin-Protein Kinase/metabolism , Primary Cell Culture , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Severity of Illness Index , Trinucleotide Repeats
6.
Oncotarget ; 8(44): 76357-76374, 2017 Sep 29.
Article in English | MEDLINE | ID: mdl-29100317

ABSTRACT

APOBEC3B, in addition to other members of the APOBEC3 gene family, has recently been intensively studied due to its identification as a gene whose activation in cancer is responsible for a specific pattern of massively occurring somatic mutations. It was recently shown that a common large deletion in the APOBEC3 cluster (the APOBEC3B deletion) may increase the risk of breast cancer. However, conflicting evidence regarding this association was also reported. In the first step of our study, using different approaches, including an in-house designed multiplex ligation-dependent probe amplification assay, we analyzed the structure of the deletion and showed that although the breakpoints are located in highly homologous regions, which may generate recurrent occurrence of similar but not identical deletions, there is no sign of deletion heterogeneity. This knowledge allowed us to distinguish transcripts of all affected genes, including the highly homologous canonical APOBEC3A and APOBEC3B, and the hybrid APOBEC3A/APOBEC3B gene. We unambiguously confirmed the presence of the hybrid transcript and showed that the APOBEC3B deletion negatively correlates with APOBEC3A and APOBEC3B expression and positively correlates with APOBEC3A/APOBEC3B expression, whose mRNA level is >10-fold and >1500-fold lower than the level of APOBEC3A and APOBEC3B, respectively. In the next step, we performed a large-scale association study in three different cohorts (2972 cases and 3682 controls) and showed no association of the deletion with breast cancer, familial breast cancer or ovarian cancer. Further, we conducted a meta-analysis that confirmed the lack of the association of the deletion with breast cancer in non-Asian populations.

7.
Oncotarget ; 7(1): 176-92, 2016 Jan 05.
Article in English | MEDLINE | ID: mdl-26484415

ABSTRACT

Somatically acquired genomic alterations that drive oncogenic cellular processes are of great scientific and clinical interest. Since the initiation of large-scale cancer genomic projects (e.g., the Cancer Genome Project, The Cancer Genome Atlas, and the International Cancer Genome Consortium cancer genome projects), a number of web-based portals have been created to facilitate access to multidimensional oncogenomic data and assist with the interpretation of the data. The portals provide the visualization of small-size mutations, copy number variations, methylation, and gene/protein expression data that can be correlated with the available clinical, epidemiological, and molecular features. Additionally, the portals enable to analyze the gathered data with the use of various user-friendly statistical tools. Herein, we present a highly illustrated review of seven portals, i.e., Tumorscape, UCSC Cancer Genomics Browser, ICGC Data Portal, COSMIC, cBioPortal, IntOGen, and BioProfiling.de. All of the selected portals are user-friendly and can be exploited by scientists from different cancer-associated fields, including those without bioinformatics background. It is expected that the use of the portals will contribute to a better understanding of cancer molecular etiology and will ultimately accelerate the translation of genomic knowledge into clinical practice.


Subject(s)
Computational Biology/methods , Data Mining/methods , Genome-Wide Association Study/methods , Genomics/methods , Neoplasms/genetics , Computational Biology/statistics & numerical data , DNA Copy Number Variations , Gene Expression Regulation, Neoplastic , Genome-Wide Association Study/statistics & numerical data , Genomics/statistics & numerical data , Humans , Mutation , Reproducibility of Results
8.
Oncotarget ; 6(27): 23399-416, 2015 Sep 15.
Article in English | MEDLINE | ID: mdl-26156018

ABSTRACT

A growing body of evidence indicates that miRNAs may be a class of genetic elements that can either drive or suppress oncogenesis. In this study we analyzed the somatic copy number variation of 14 miRNA genes frequently found to be either over- or underexpressed in lung cancer, as well as two miRNA biogenesis genes, DICER1 and DROSHA, in non-small-cell lung cancer (NSCLC). Our analysis showed that most analyzed miRNA genes undergo substantial copy number alteration in lung cancer. The most frequently amplified miRNA genes include the following: miR-30d, miR-21, miR-17 and miR-155. We also showed that both DICER1 and DROSHA are frequently amplified in NSCLC. The copy number variation of DICER1 and DROSHA correlates well with their expression and survival of NSCLC and other cancer patients. The increased expression of DROSHA and DICER1 decreases and increases the survival, respectively. In conclusion, our results show that copy number variation may be an important mechanism of upregulation/downregulation of miRNAs in cancer and suggest an oncogenic role for DROSHA.


Subject(s)
Carcinoma, Non-Small-Cell Lung/metabolism , DEAD-box RNA Helicases/metabolism , Gene Expression Regulation, Neoplastic , Lung Neoplasms/metabolism , MicroRNAs/metabolism , Ribonuclease III/metabolism , Adult , Aged , Aged, 80 and over , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/mortality , Chromosomes, Human, Pair 5/genetics , Down-Regulation , Female , Gene Dosage , Gene Expression Profiling , Humans , Lung Neoplasms/genetics , Lung Neoplasms/mortality , Male , Middle Aged , Retrospective Studies , Treatment Outcome , Up-Regulation
9.
Sci Rep ; 5: 10424, 2015 May 21.
Article in English | MEDLINE | ID: mdl-25994375

ABSTRACT

Only approximately 50% of all familial breast cancers can be explained by known genetic factors, including mutations in BRCA1 and BRCA2. One of the most extensively studied candidates for breast and/or ovarian cancer susceptibility is BARD1. Although it was suggested that large mutations may contribute substantially to the deleterious variants of BARD1, no systematic study of the large mutations in BARD1 has been performed. To further elucidate the role of large mutations in BARD1, we designed a multiplex ligation-dependent probe amplification (MLPA) assay and performed an analysis of 504 women with a familial breast and/or ovarian cancer and 313 patients with ovarian cancer. The investigation did not reveal any large mutations in the BARD1 gene. Although the analysis was not focused on identification of small mutations, we detected seven deleterious or potentially deleterious point mutations, which contribute substantially to the total number of BARD1 mutations detected so far. In conclusion, although we cannot exclude the presence of large mutations in BARD1, our study indicates that such mutations do not contribute substantially to the risk of breast and/or ovarian cancer. However, it has to be noted that our results may be specific to the Polish population.


Subject(s)
Ovarian Neoplasms/genetics , Tumor Suppressor Proteins/genetics , Ubiquitin-Protein Ligases/genetics , White People/genetics , Base Sequence , Breast Neoplasms/genetics , Breast Neoplasms/pathology , DNA Mutational Analysis , Female , Humans , Multiplex Polymerase Chain Reaction , Mutation, Missense , Ovarian Neoplasms/pathology , Poland , Polymorphism, Single Nucleotide
10.
PLoS One ; 10(2): e0117983, 2015.
Article in English | MEDLINE | ID: mdl-25719557

ABSTRACT

Lung cancer is the leading cause of cancer-related death worldwide. Recent progress in lung cancer diagnosis and treatment has been achieved due to a better understanding the molecular mechanisms of the disease and the identification of biomarkers that allow more specific cancer treatments. One of the best known examples of personalized therapy is the use of tyrosine kinase inhibitors, such as gefitinib and erlotinib, for the successful treatment of non-small-cell lung cancer patients selected based on the specific EGFR mutations. Therefore, the reliable detection of mutations is critical for the application of appropriate therapy. In this study, we tested a two-tiered mutation detection strategy using real-time PCR assays as a well-validated high-sensitivity method and multiplex ligation-dependent probe amplification (MLPA)-based EGFRmut+ assay as a second-tier standard-sensitivity method. One additional advantage of the applied MLPA method is that it allows the simultaneous detection of EGFR mutations and copy-number alterations (i.e., amplifications) in EGFR, MET and ERBB2. Our analysis showed high concordance between these two methods. With the use of this two-tier strategy, we reliably determined the frequency of EGFR mutations and EGFR, MET and ERBB2 amplifications in over 200 lung cancer samples. Additionally, taking advantage of simultaneous copy number and small mutation analyses, we showed a very strong correlation between EGFR mutations and EGFR amplifications and a mutual exclusiveness of EGFR mutations/amplifications with MET and ERBB2 amplifications. Our results proved the reliability and usefulness of the two-tiered EGFR testing strategy.


Subject(s)
Biomarkers, Tumor/genetics , Early Detection of Cancer/methods , ErbB Receptors/genetics , Gene Amplification , Lung Neoplasms/diagnosis , Multiplex Polymerase Chain Reaction/methods , Point Mutation , Adult , Aged , Aged, 80 and over , Humans , Lung Neoplasms/genetics , Middle Aged
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