Alternative p38MAPKs as biomarkers in the interplay of colon cancer and inflammatory bowel diseases

Trabajo presentado en el 44º Congreso Nacional de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Málaga (España) del 06 al 09 de septiembre de 2022.Chronic inflammation in inflammatory bowel disease (IBD) is a risk factor for Colorectal cancer (CRC) development, but our understanding of this interplay at a molecular level is still limited. p38γ and p38δ, are central in the development of mouse colitis-associated CRC (CAC) by modulating the inflammatory immune response. However, their implication in human CRC and IBD is not well defined. In this study we perform an integrative analysis of p38γ and p38δ mRNA and protein expression and activation in human patients; using human CRC derived organoids and plasma samples, as well as data from different human CRC and IBD mRNA databases. We found that, p38δ levels were decreased, whereas p38γ expression and phosphorylation were significantly increased in CRC compared to normal colon samples. This increase correlated with the expression of genes implicated in inflammation. Examine of p38γ/p38δ in IBD patients showed that p38γ mRNA and protein levels were increased in Crohn’s disease and ulcerative colitis patients. Contrary, p38δ mRNA was significantly decreased. We also investigated the expression of miRNAs, miR-128-2, miR133a and miR-155, implicated in inflammation and cancer development. In mouse model of colitis and CAC, miR128-2 level was regulated by p38γ/p38δ. In the plasma of IBD and CRC patients, miR128-2 was increased compared to healthy donors, and this correlated with p38γ and p38δ levels. Our results show an opposite regulation of p38γ and p38δ in both CRC and IBD; and suggest that p38γ acts as a link between colitis and CRC by favouring an inflammatory environment that promotes tumour development. We provided evidence that p38γ/p38δ, together with miR-128-2, can be useful as biomarkers, and as potential treatment targets, for colitis and early-stage CRC

p38γ and p38δ as biomarkers in the interplay of colon cancer and inflammatory bowel diseases

descripción no proporcionada por scopusThis research was funded by the MCIN/AEI/10.13039/501100011033 (PID2019-108349RB100 and SAF2016-79792R) to AC and JJSE; Villum Foundation, grant no. 13152 to KA; by Agencia Estatal de Investigación (PID2019-104867RBI00/AEI/10.13039/501100011033) and the Instituto de Salud Carlos III- Fondo Europeo de Desarrollo Regional (CIBERONC/CB16/12/00273 and ICI20/00057) to AM and AB. PF received MCIN FPI fellowship (BES-2017-080139)

Precision Nucleic Acid Diagnostics using Novel Oligonucleotide Reagents and Amplification-Free Assays

Precision in cancer diagnostics is of high interest to improve survival rates, costs and treatment guidance. Nucleic acid biomarkers are promising tools for use in precision cancer diagnostics. However, detection of nucleic acid biomarkers remains a challenge. This thesis will address some of the issues related to detection of nucleic acid biomarkers for early cancer diagnostic.In the thesis, three strategies to optimize current amplification-free FLEET platform, established in Astakhova group, have been investigated. We applied calculations from the Peyrard-Bishop model to make insertions of LNA in high-affinity capture sequences targeting oncogenes, and predicted their melting temperatures (Tm). Selected probes were synthesized, and had their Tm measured, resulting in an accuracy of 1°C. This indicated that the PB model can be applied for future design of ultra-specific probes. Secondly, we implemented the FLEET assay in microfluidic PMMA chips using TC-tagged capture probes immobilized with UV light. We fabricated microfluidic PMMA chips and estimated LOD to be 6.4pM – 32pM using a spectrofluorometer.The assay was tested with BRAF-probes, designed with the PB model. This showed a detectable signal, but needs to be optimized.We describe a quantitative detection method for mutated microRNA in human plasma samples. Specific oligonucleotides designed from a Peyrard-Bishop model allowed accurate prediction of target:probe recognition affinity and specificity. The assay allowed identification of singlenucleotide polymorphism mismatch profiles in clinically relevant microRNA-128-2-3p, showing terminal mutations that correlate positively with inflammatory colitis and colorectal cancer. Related to production of probes, we describe a new approach to make pools of microRNA targeting breast cancer cells. The microRNA pools were synthesized at once on the same solid-support using ‘Tandem Oligonucleotide Synthesis’ strategy. We make up to four consecutive microRNA using 2’/3’OAc nucleotide phosphoramidites. The developed phosphoramidites combined give a cleavable moiety that separates the microRNAs and are cleaved using standard cleavage conditions. Furthermore, we investigate making branched pools (microRNA dendrimers) versus linear pools as a strategy to further improve the product yields. Our approach provides microRNA pools in high yields, which is of relevance to the growing demand on synthetic RNA oligomers for nucleic acid research and technology. Targeted sequencing has great importance in finding new variants of nucleic acid biomarkers. In this thesis, we optimize Oligonucleotide-Selective Sequencing, established in the Ji Research lab. We investigate the effect of primer spacing using synthesized branched oligonucleotide as primers, with the aim of improving yield and sensitivity. The branching primers were found to improve sensitivity by 2-fold compared to control primers which is promising

MicroRNA Pools Synthesized Using Tandem Solid-Phase Oligonucleotide Synthesis

Herein, we describe a new approach to make pools of microRNA targeting breast cancer cells. The microRNA pools were synthesized at once on the same solid support using the “Tandem Oligonucleotide Synthesis” strategy. We make up to four consecutive microRNAs (miR129-1-5p, miR31, miR206, and miR27b-3p) using 2′/3′OAc nucleotide phosphoramidites, with the total length of the pool reaching 88 nucleotides. The developed phosphoramidites, when combined, give a cleavable moiety that separates the microRNAs and is cleaved using standard post-RNA synthesis cleavage conditions. Furthermore, we investigate making branched pools (microRNA dendrimers) versus linear pools as a strategy to further improve the product yields. Our approach provides with microRNA pools in high yields, which is of relevance to the growing demand on synthetic RNA oligomers for nucleic acid research and technology.</p

Mutations in microRNA-128-2-3p identified with amplification-free hybridization assay.

We describe a quantitative detection method for mutated microRNA in human plasma samples. Specific oligonucleotides designed from a Peyrard-Bishop model allowed accurate prediction of target:probe recognition affinity and specificity. Our amplification-free tandem bead-based hybridization assay had limit of detection of 2.2 pM. Thereby, the assay allowed identification of single-nucleotide polymorphism mismatch profiles in clinically relevant microRNA-128-2-3p, showing terminal mutations that correlate positively with inflammatory colitis and colorectal cancer

New Approaches to Moderate CRISPR-Cas9 Activity:Addressing Issues of Cellular Uptake and Endosomal Escape

CRISPR-Cas9 is rapidly entering molecular biology and biomedicine as a promising gene-editing tool. A unique feature of CRISPR-Cas9 is a single-guide RNA directing a Cas9 nuclease toward its genomic target. Herein, we highlight new approaches for improving cellular uptake and endosomal escape of CRISPR-Cas9. As opposed to other recently published works, this review is focused on non-viral carriers as a means to facilitate the cellular uptake of CRISPR-Cas9 through endocytosis. The majority of non-viral carriers, such as gold nanoparticles, polymer nanoparticles, lipid nanoparticles, and nanoscale zeolitic imidazole frameworks, is developed with a focus toward optimizing the endosomal escape of CRISPR-Cas9 by taking advantage of the acidic environment in the late endosomes. Among the most broadly used methods for in vitro and ex vivo ribonucleotide protein transfection are electroporation and microinjection. Thus, other delivery formats are warranted for in vivo delivery of CRISPR-Cas9. Herein, we specifically revise the use of peptide and nanoparticle-based systems as platforms for CRISPR-Cas9 delivery in vivo. Finally, we highlight future perspectives of the CRISPR-Cas9 gene-editing tool and the prospects of using non-viral vectors to improve its bioavailability and therapeutic potential