Validation of miRNA-mRNA interactions by electrophoretic mobility shift assays

BACKGROUND: MicroRNAs are small non-coding RNAs involved in gene expression regulation by targeting specific regions in the 3[prime]-UTR of the mRNA of their target genes. This binding leads to a decrease in the protein levels of such genes either by mRNA degradation or mRNA destabilization and translation inhibition. The interaction between a miRNA and its target mRNAs is usually studied by co-transfection of a reporter expression vector containing the 3[prime]-UTR region of the mRNA and an inhibitory or precursor molecule for the miRNA. This approach, however, does not measure the direct and physical interaction between a miRNA and a specific mRNA. FINDINGS: RNA molecules corresponding to miR-224 and to the 3[prime]-UTR of SLC4A4 were incubated together and their interaction studied under different binding conditions using electrophoretic mobility shift assays. A direct and specific interaction between miR-224 and SLC4A4 mRNA was observed. This interaction was abolished in the presence of competitors. CONCLUSIONS: In this study, we explored a new application for the electrophoretic mobility shift assay and we demonstrated that it is a useful alternative method to assess, in a direct and specific manner, whether a miRNA binds to a specific predicted target mRNA

The Molecular Tumor Board Portal supports clinical decisions and automated reporting for precision oncology

Cancer; Cancer geneticsCáncer; Genética del cáncerCàncer; Genètica del càncerThere is a growing need for systems that efficiently support the work of medical teams at the precision-oncology point of care. Here, we present the implementation of the Molecular Tumor Board Portal (MTBP), an academic clinical decision support system developed under the umbrella of Cancer Core Europe that creates a unified legal, scientific and technological platform to share and harness next-generation sequencing data. Automating the interpretation and reporting of sequencing results decrease the need for time-consuming manual procedures that are prone to errors. The adoption of an expert-agreed process to systematically link tumor molecular profiles with clinical actions promotes consistent decision-making and structured data capture across the connected centers. The use of information-rich patient reports with interactive content facilitates collaborative discussion of complex cases during virtual molecular tumor board meetings. Overall, streamlined digital systems like the MTBP are crucial to better address the challenges brought by precision oncology and accelerate the use of emerging biomarkers.Open access funding provided by Karolinska Institut

Support systems to guide clinical decision-making in precision oncology: The Cancer Core Europe Molecular Tumor Board Portal.

To the editor: the optimal management of cancer patients is increasingly dependent on individualized treatments guided by tumor sequencing data. As comprehensive genomic tests become routine in many disease settings and academic centers promote omics-guided clinical trial recruitment, accurate and scalable data interpretation represents a major challenge. The meticulous task of matching tumor alterations with approved or experimental therapies relies heavily on the expertise of each center

Overexpression of S100A4 in human cancer cell lines resistant to methotrexate

Methotrexate is a chemotherapeutic drug that is used in therapy of a wide variety of cancers. The efficiency of treatment with this drug is compromised by the appearance of resistance. Combination treatments of MTX with other drugs that could modulate the expression of genes involved in MTX resistance would be an adequate strategy to prevent the development of this resistance. Methods: The differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. A global comparison of all the studied cell lines was performed in order to find out differentially expressed genes in the majority of the MTX-resistant cells. S100A4 mRNA and protein levels were determined by RT-Real-Time PCR and Western blot, respectively. Functional validations of S100A4 were performed either by transfection of an expression vector for S100A4 or a siRNA against S100A4. Transfection of an expression vector encoding for β-catenin was used to inquire for the possible transcriptional regulation of S100A4 through the Wnt pathway. Results: S100A4 is overexpressed in five out of the seven MTX-resistant cell lines studied. Ectopic overexpression of this gene in HT29 sensitive cells augmented both the intracellular and extracellular S100A4 protein levels and caused desensitization toward MTX. siRNA against S100A4 decreased the levels of this protein and caused a chemosensitization in combined treatments with MTX. β-catenin overexpression experiments support a possible involvement of the Wnt signaling pathway in S100A4 transcriptional regulation in HT29 cells. Conclusions: S100A4 is overexpressed in many MTX-resistant cells. S100A4 overexpression decreases the sensitivity of HT29 colon cancer human cells to MTX, whereas its knockdown causes chemosensitization toward MTX. Both approaches highlight a role for S100A4 in MTX resistanc

The Molecular Tumor Board Portal supports clinical decisions and automated reporting for precision oncology.

There is a growing need for systems that efficiently support the work of medical teams at the precision-oncology point of care. Here, we present the implementation of the Molecular Tumor Board Portal (MTBP), an academic clinical decision support system developed under the umbrella of Cancer Core Europe that creates a unified legal, scientific and technological platform to share and harness next-generation sequencing data. Automating the interpretation and reporting of sequencing results decrease the need for time-consuming manual procedures that are prone to errors. The adoption of an expert-agreed process to systematically link tumor molecular profiles with clinical actions promotes consistent decision-making and structured data capture across the connected centers. The use of information-rich patient reports with interactive content facilitates collaborative discussion of complex cases during virtual molecular tumor board meetings. Overall, streamlined digital systems like the MTBP are crucial to better address the challenges brought by precision oncology and accelerate the use of emerging biomarkers

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Polypurine Reverse Hoogsteen hairpins: stability, lack of immunogenicity and gene silencing in cancer therapy

[spa] Este trabajo se centra en el estudio de la estabilidad y potencial inmunogénico de las moléculas llamadas PPRHs (Polypurine Reverse Hoogsteen hairpins), y en su uso como herramienta de silenciamiento génico. Los PPRHs son moléculas de DNA no modificadas. Están formadas por dos cadenas antiparalelas de polipurinas unidas por un bucle pentatimidínico, lo que permite la formación intramolecular de enlaces de Hoogsteen reversos entre ambas cadenas. Anteriormente se demostró que los PPRH son capaces de unirse a una secuencia de dsDNA mediante enlaces de Watson-Crick, formando un triplex que desplaza la cadena polipurínica de la diana. La prueba de principio para el uso de los PPRH como agente terapéuticos se llevó a cabo con un PPRH dirigido al gen de la survivina el cual fue validado tanto in vitro como in vivo. En esta tesis se ha profundizado en el conocimiento de los PPRHs. Se ha establecido que estas moléculas son estables en diferentes tipos de suero, así como intracelularmente. Además se determinó que los PPRHs, no inducen la activación de la respuesta inmune innata, ya que su transfección a una línea monocítica no incrementó los niveles de expresión de los factores de transcripción IRF3 y NF-κB, y tampoco de las citoquinas proinflamatorias IL-6, TNF-α, IFN-α, IFN-β, IL-1β, y IL-18. Además, los PPRHs no indujeron la activación del inflamasoma. También se diseñaron dos estructuras nuevas de PPRH: PPRH circulares y PPRHs basados en RNA, y se estudiaron diversos parámetros como su capacidad de unión a la diana y su citotoxicidad. Para ampliar la aplicabilidad de estas moléculas, se validó el uso de los PPRHs sobre diversos genes relevantes en cáncer: BCL2, MDM2, MYC, TOP1 y MTOR. Esta validación se realizó en diversas líneas tumorales (PC3, MIA PaCa-2, HCT116, SKBR3, MCF7 y MDA-MB-468) observando que los PPRHs fueron capaces de disminuir la supervivencia de las células y la expresión de los genes diana, y de aumentar la apoptosis. Finalmente, se realizó una aproximación para incrementar la especificidad de los PPRHs, la cual incluye el uso de un aptámero de DNA dirigido a la proteína de membrana HER2.[eng] This work is focused on the study of the stability and immunogenic properties of the Polypurine Reverse Hoogsteen hairpins (PPRHs), and on their use as a gene-silencing tool. PPRHs are non-modified DNA molecules formed by two antiparallel polypurine strands linked by a pentathymidine loop that allows the formation of intramolecular reverse Hoogsteen bonds between both strands. Previously in our laboratory it was demonstrated that these hairpins bind to their polypyrimidine target in a dsDNA via Watson-Crick bonds, displacing the polypurine strand of the target duplex. The effect of PPRHs in cells and their mechanism of action were first described using PPRHs designed against the template and coding strands of the DHFR gene. A PPRH against survivin was further validated in a xenograft tumor model, establishing the proof of principle for the use of PPRHs as a therapeutic tool. In this work we increased the knowledge we have about PPRHs. We were able to establish that PPRHs, unlike siRNAs, are very stable molecules in different types of serum and inside the cells. We also established that PPRHs do not induce the innate immune response, since they do not induce the levels of neither the transcription factors IRF3 and NF-κB, nor the proinflammatory cytokines IL-6, TNF-α, IFN-α, IFN-β, IL-1β, and IL-18. Additionally, unlike siRNAs, PPRHs did not trigger the activation of the inflammasome. Another element that we studied was the modification of the PPRH structure, since it has been shown that circular structures can provide advantages over linear structures. Therefore, we analyzed the efficacy of two other types of PPRH: i) nicked-circle-PPRHs, a new structure in which a second loop was introduced to form a nearly circular sequence, and ii) PPRHs made out of RNA (RNA-PPRHs). To broaden the applicability of PPRHs in cancer therapy, we evaluated their capacity to silence genes involved in a variety of biological functions linked to cancer hallmarks. The genes selected were: BCL2, MDM2, MYC, TOP1 and MTOR, and the validation of the PPRHs was performed in different cancer cells lines (PC3, MIA PaCa2, HCT116, SKBR3, MCF7 and MDA-MB-468). Regardless of the gene or cell line tested, PPRHs were able to decrease cell survival and mRNA expression levels, and to increase apoptosis, to a greater or lesser extent. Finally, we also present an approach to increase the specificity of PPRHs that involves the use of a DNA aptamer that has been shown to have an effect in HER2 positve cells

Overexpression of S100A4 in human cancer cell lines resistant to methotrexate

Abstract Background Methotrexate is a chemotherapeutic drug that is used in therapy of a wide variety of cancers. The efficiency of treatment with this drug is compromised by the appearance of resistance. Combination treatments of MTX with other drugs that could modulate the expression of genes involved in MTX resistance would be an adequate strategy to prevent the development of this resistance. Methods The differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. A global comparison of all the studied cell lines was performed in order to find out differentially expressed genes in the majority of the MTX-resistant cells. S100A4 mRNA and protein levels were determined by RT-Real-Time PCR and Western blot, respectively. Functional validations of S100A4 were performed either by transfection of an expression vector for S100A4 or a siRNA against S100A4. Transfection of an expression vector encoding for β-catenin was used to inquire for the possible transcriptional regulation of S100A4 through the Wnt pathway. Results S100A4 is overexpressed in five out of the seven MTX-resistant cell lines studied. Ectopic overexpression of this gene in HT29 sensitive cells augmented both the intracellular and extracellular S100A4 protein levels and caused desensitization toward MTX. siRNA against S100A4 decreased the levels of this protein and caused a chemosensitization in combined treatments with MTX. β-catenin overexpression experiments support a possible involvement of the Wnt signaling pathway in S100A4 transcriptional regulation in HT29 cells. Conclusions S100A4 is overexpressed in many MTX-resistant cells. S100A4 overexpression decreases the sensitivity of HT29 colon cancer human cells to MTX, whereas its knockdown causes chemosensitization toward MTX. Both approaches highlight a role for S100A4 in MTX resistance.</p