EGCG Disrupts the LIN28B/Let-7 Interaction and Reduces Neuroblastoma Aggressiveness.

Neuroblastoma (NB) is the most commonly diagnosed extracranial solid tumor in children, accounting for 15% of all childhood cancer deaths. Although the 5-year survival rate of patients with a high-risk disease has increased in recent decades, NB remains a challenge in pediatric oncology, and the identification of novel potential therapeutic targets and agents is an urgent clinical need. The RNA-binding protein LIN28B has been identified as an oncogene in NB and is associated with a poor prognosis. Given that LIN28B acts by negatively regulating the biogenesis of the tumor suppressor let-7 miRNAs, we reasoned that selective interference with the LIN28B/let-7 miRNA interaction would increase let-7 miRNA levels, ultimately leading to reduced NB aggressiveness. Here, we selected (-)-epigallocatechin 3-gallate (EGCG) out of 4959 molecules screened as the molecule with the best inhibitory activity on LIN28B/let-7 miRNA interaction and showed that treatment with PLC/PLGA-PEG nanoparticles containing EGCG (EGCG-NPs) led to an increase in mature let-7 miRNAs and a consequent inhibition of NB cell growth. In addition, EGCG-NP pretreatment reduced the tumorigenic potential of NB cells in vivo. These experiments suggest that the LIN28B/let-7 miRNA axis is a good therapeutic target in NB and that EGCG, which can interfere with this interaction, deserves further preclinical evaluation.

The DNA-repair protein APE1 participates with hnRNPA2B1 to motif-enriched and prognostic miRNA secretion.

The base excision repair (BER) Apurinic/apyrimidinic endonuclease 1 (APE1) enzyme is endowed with several non-repair activities including miRNAs processing. APE1 is overexpressed in many cancers but its causal role in the tumorigenic processes is largely unknown. We recently described that APE1 can be actively secreted by mammalian cells through exosomes. However, APE1 role in EVs or exosomes is still unknown, especially regarding a putative regulatory function on vesicular small non-coding RNAs. Through dedicated transcriptomic analysis on cellular and vesicular small RNAs of different APE1-depleted cancer cell lines, we found that miRNAs loading into EVs is a regulated process, dependent on APE1, distinctly conveying RNA subsets into vesicles. We identified APE1-dependent secreted miRNAs characterized by enriched sequence motifs and possible binding sites for APE1. In 33 out of 34 APE1-dependent-miRNA precursors, we surprisingly found EXO-motifs and proved that APE1 cooperates with hnRNPA2B1 for the EV-sorting of a subset of miRNAs, including miR-1246, through direct binding to GGAG stretches. Using TCGA-datasets, we showed that these miRNAs identify a signature with high prognostic significance in cancer. In summary, we provided evidence that the ubiquitous DNA-repair enzyme APE1 is part of the EV protein cargo with a novel post-transcriptional role for this ubiquitous DNA-repair enzyme that could explain its role in cancer progression. These findings could open new translational perspectives in cancer biology.

Shaping the future of oral cancer diagnosis: advances in salivary proteomics.

INTRODUCTION: Saliva has gained increasing attention in the quest for disease biomarkers. Because it is a biological fluid that can be collected is an easy, painless, and safe way, it has been increasingly studied for the identification of oral cancer biomarkers. This is particularly important because oral cancer is often diagnosed at late stages with a poor prognosis. AREAS COVERED: The review addresses the evolution of the experimental approaches used in salivary proteomics studies of oral cancer over the years and outlines advantages and pitfalls related to each one. In addition, examines the current landscape of oral cancer biomarker discovery and translation focusing on salivary proteomic studies. This discussion is based on an extensive literature search (PubMed, Scopus and Google Scholar). EXPERT OPINION: The introduction of mass spectrometry has revolutionized the study of salivary proteomics. In the future, the focus will be on refining existing methods and introducing powerful experimental techniques such as mass spectrometry with selected reaction monitoring, which, despite their effectiveness, are still underutilized due to their high cost. In addition, conducting studies with larger cohorts and establishing standardized protocols for salivary proteomics are key challenges that need to be addressed in the coming years.

Revisiting Multi-Omics Data to Unravel Galectins as Prognostic Factors in Head and Neck Squamous Cell Carcinoma.

Head and Neck Squamous Cell Carcinoma (HNSCC) is a malignant cancer with a poor prognosis. Galectins (Gal) have been the subject of intensive research, but the comparative prognostic value of each Gal type is not yet understood. Therefore, a literature search for evaluating galectins as prognostic biomarkers in HNSCC was conducted. The relationship between Gal expression in HNSCC with HPV and TP53 mutational status was assessed using the UALCAN database. The impact of these biomarkers on prognosis was analyzed using ToPP and CPPA web tools. The expression of galectins in the tumor microenvironment and the impact on prognosis depending on the cancer immune subtype were analyzed using single-cell RNA sequencing. Gal-1 and Gal-3BP were shown to be promising biomarkers with a triple function for the prediction of HPV and TP53 mutational status, stratification of the HNSCC prognosis, and prediction of the response to treatment. In addition, these two galectins have been shown to be most influenced by the tumor microenvironment of HNSCC. Gal-1 and Gal-3BP are the most promising galectins in HNSCC. Furthermore, this study highlights the need for further studies to evaluate galectins in HNSCC and clarify the role of individual Gals in the patient's stratification.

International Society for Extracellular Vesicles Workshop. QuantitatEVs: multiscale analyses, from bulk to single extracellular vesicle.

The 'QuantitatEVs: multiscale analyses, from bulk to single vesicle' workshop aimed to discuss quantitative strategies and harmonized wet and computational approaches toward the comprehensive analysis of extracellular vesicles (EVs) from bulk to single vesicle analyses with a special focus on emerging technologies. The workshop covered the key issues in the quantitative analysis of different EV-associated molecular components and EV biophysical features, which are considered the core of EV-associated biomarker discovery and validation for their clinical translation. The in-person-only workshop was held in Trento, Italy, from January 31st to February 2nd, 2023, and continued in Milan on February 3rd with "Next Generation EVs", a satellite event dedicated to early career researchers (ECR). This report summarizes the main topics and outcomes of the workshop.

Integrating extracellular vesicle and circulating cell-free DNA analysis using a single plasma aliquot improves the detection of HER2 positivity in breast cancer patients.

Multi-analyte liquid biopsies represent an emerging opportunity for non-invasive cancer assessment. We developed ONCE (ONe Aliquot for Circulating Elements), an approach for the isolation of extracellular vesicles (EV) and cell-free DNA (cfDNA) from a single aliquot of blood. We assessed ONCE performance to classify HER2-positive early-stage breast cancer (BrCa) patients by combining EV-associated RNA (EV-RNA) and cfDNA signals on n=64 healthy donors (HD) and non-metastatic BrCa patients. Specifically, we isolated EV-enriched samples by a charge-based (CB) method and investigated EV-RNA and cfDNA by next-generation sequencing (NGS) and by digital droplet PCR (ddPCR). Sequencing of cfDNA and EV-RNA from HER2- and HER2+ patients demonstrated concordance with in situ molecular analyses of matched tissues. Combined analysis of the two circulating analytes by ddPCR showed increased sensitivity in ERBB2/HER2 detection compared to single nucleic acid components. Multi-analyte liquid biopsy prediction performance was comparable to tissue-based sequencing results from TCGA. Also, imaging flow cytometry analysis revealed HER2 protein on the surface of EV isolated from the HER2+ BrCa plasma, thus corroborating the potential relevance of studying EV as companion analyte to cfDNA. This data confirms the relevance of combining cfDNA and EV-RNA for HER2 cancer assessment and supports the ONCE as a valuable tool for multi-analytes liquid biopsies' clinical implementation.

Reevaluation of bromodomain ligands targeting BAZ2A.

BAZ2A promotes migration and invasion in prostate cancer. Two chemical probes, the specific BAZ2-ICR, and the BAZ2/BRD9 cross-reactive GSK2801, interfere with the recognition of acetylated lysines in histones by the bromodomains of BAZ2A and of its BAZ2B paralog. The two chemical probes were tested in prostate cancer cell lines with opposite androgen susceptibility. BAZ2-ICR and GSK2801 showed different cellular efficacies in accordance with their unequal selectivity profiles. Concurrent inhibition of BAZ2 and BRD9 did not reproduce the effects observed with GSK2801, indicating possible off-targets for this chemical probe. On the other hand, the single BAZ2 inhibition by BAZ2-ICR did not phenocopy genetic ablation, demonstrating that bromodomain interference is not sufficient to strongly affect BAZ2A functionality and suggesting a PROTAC-based chemical ablation as an alternative optimization strategy and a possible therapeutic approach. In this context, we also present the crystallographic structures of BAZ2A in complex with the above chemical probes. Binding poses of TP-238 and GSK4027, chemical probes for the bromodomain subfamily I, and two ligands of the CBP/EP300 bromodomains identify additional headgroups for the development of BAZ2A ligands.

Identification of a BAZ2A-Bromodomain Hit Compound by Fragment Growing.

BAZ2A is an epigenetic regulator affecting transcription of ribosomal RNA. It is overexpressed in aggressive and recurrent prostate cancer, promoting cellular migration. Its bromodomain is characterized by a shallow and difficult-to-drug pocket. Here, we describe a structure-based fragment-growing campaign for the identification of ligands of the BAZ2A bromodomain. By combining docking, competition binding assays, and protein crystallography, we have extensively explored the interactions of the ligands with the rim of the binding pocket, and in particular ionic interactions with the side chain of Glu1820, which is unique to BAZ2A. We present 23 high-resolution crystal structures of the holo BAZ2A bromodomain and analyze common bromodomain/ligand motifs and favorable intraligand interactions. Binding of some of the compounds is enantiospecific, with affinity in the low micromolar range. The most potent ligand has an equilibrium dissociation constant of 7 µM and a good selectivity over the paralog BAZ2B bromodomain.

Identification and Characterization of an RRM-Containing, RNA Binding Protein in Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative pathogen, known to acquire resistance to antibiotics used in the clinic. The RNA-binding proteome of this bacterium is poorly characterized, in particular for what concerns the proteins containing RNA Recognition Motif (RRM). Here, we browsed the A. baumannii proteome for homologous proteins to the human HuR(ELAVL1), an RNA binding protein containing three RRMs. We identified a unique locus that we called AB-Elavl, coding for a protein with a single RRM with an average of 34% identity to the first HuR RRM. We also widen the research to the genomes of all the bacteria, finding 227 entries in 12 bacterial phyla. Notably we observed a partial evolutionary divergence between the RNP1 and RNP2 conserved regions present in the prokaryotes in comparison to the metazoan consensus sequence. We checked the expression at the transcript and protein level, cloned the gene and expressed the recombinant protein. The X-ray and NMR structural characterization of the recombinant AB-Elavl revealed that the protein maintained the typical ß1α1ß2ß3α2ß4 and three-dimensional organization of eukaryotic RRMs. The biochemical analyses showed that, although the RNP1 and RNP2 show differences, it can bind to AU-rich regions like the human HuR, but with less specificity and lower affinity. Therefore, we identified an RRM-containing RNA-binding protein actually expressed in A. baumannii.

Expression of Cellular and Extracellular TERRA, TERC and TERT in Hepatocellular Carcinoma.

Non-coding RNAs are transcribed from telomeres and the telomeric repeat-containing RNAs (TERRA) are implicated in telomere homeostasis and in cancer. In this study, we aimed to assess in hepatocellular carcinoma (HCC) the cellular and extracellular expression of TERRA, the telomerase RNA subunit (TERC) and the telomerase catalytic subunit (TERT). We determined by qPCR the expression level of TERRA 1_2_10_13q, TERRA 15q, TERRA XpYp, TERC and of TERT mRNA in HCC tissues and in the plasma of HCC patients. Further, we profiled the same transcripts in the HCC cell lines, HA22T/VGH and SKHep1C3, and in the extracellular vesicles (EVs) derived from their secretomes. We found that the expression of TERRA and TERT mRNA was significantly deregulated in HCC, being TERRA downregulated and TERT mRNA upregulated in HCC tissues vs. the peritumoral (PT) ones, and the receiver operating characteristic (ROC) curve analyses revealed a significant ability in discriminating HCC from PT tissue. Further, the determinations of circulating TERRA and TERC showed higher amounts of these transcripts in the plasma of HCC patients vs. controls and ROC analyses gave significant results. The expression characterization of the cultured HCC cells showed their ability to produce and secrete TERRA and TERC into the EVs; the ability to produce TERT mRNA that was not detectable in the EVs; and the ability to respond to sorafenib treatment increasing TERRA expression. Our results highlight that: (i) both cellular and extracellular expressions of TERRA and TERC are dysregulated in HCC as well as the cellular expression of TERT mRNA and (ii) the combined detection of TERRA and TERC in plasma may represent a promising approach for non-invasive diagnostic molecular indicators of HCC.

Zebrafish Melanoma-Derived Interstitial EVs Are Carriers of ncRNAs That Induce Inflammation.

Extracellular vesicles (EVs) are membranous particles released by all cell types. Their role as functional carrier of bioactive molecules is boosted by cells that actively secrete them in biological fluids or in the intercellular space (interstitial EVs, iEVs). Here we have optimised a method for the isolation and characterization of zebrafish iEVs from whole melanoma tissues. Zebrafish melanoma iEVs are around 140 nm in diameter, as determined by nanoparticle tracking and transmission electron microscopy (TEM) analysis. Western blot analysis shows enrichment for CD63 and Alix in the iEV fraction, but not in melanoma cell lysates. Super resolution and confocal microscopy reveal that purified zebrafish iEVs are green fluorescent protein positive (GFP+), indicating that they integrate the oncogene GFP-HRASV12G used to induce melanoma in this model within their vesicular membrane or luminal content. Analysis of RNA-Seq data found 118 non-coding (nc)RNAs differentially distributed between zebrafish melanoma and their iEVs, with only 17 of them being selectively enriched in iEVs. Among these, the RNA components of RNAses P and MRP, which process ribosomal RNA precursors, mitochondrial RNAs, and some mRNAs, were enriched in zebrafish and human melanoma EVs, but not in iEVs extracted from brain tumours. We found that melanoma iEVs induce an inflammatory response when injected in larvae, with increased expression of interferon responsive genes, and this effect is reproduced by MRP- or P-RNAs injected into circulation. This suggests that zebrafish melanoma iEVs are a source of MRP- and P-RNAs that can trigger inflammation in cells of the innate immune system.

C9orf72 ALS/FTD dipeptide repeat protein levels are reduced by small molecules that inhibit PKA or enhance protein degradation.

Intronic GGGGCC (G4C2) hexanucleotide repeat expansion within the human C9orf72 gene represents the most common cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of repeat-containing C9orf72 RNA results in the production of neurotoxic dipeptide-repeat proteins (DPRs). Here, we developed a high-throughput drug screen for the identification of positive and negative modulators of DPR levels. We found that HSP90 inhibitor geldanamycin and aldosterone antagonist spironolactone reduced DPR levels by promoting protein degradation via the proteasome and autophagy pathways respectively. Surprisingly, cAMP-elevating compounds boosting protein kinase A (PKA) activity increased DPR levels. Inhibition of PKA activity, by both pharmacological and genetic approaches, reduced DPR levels in cells and rescued pathological phenotypes in a Drosophila model of C9ALS/FTD. Moreover, knockdown of PKA-catalytic subunits correlated with reduced translation efficiency of DPRs, while the PKA inhibitor H89 reduced endogenous DPR levels in C9ALS/FTD patient-derived iPSC motor neurons. Together, our results suggest new and druggable pathways modulating DPR levels in C9ALS/FTD.

Decoding distinctive features of plasma extracellular vesicles in amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a multifactorial, multisystem motor neuron disease for which currently there is no effective treatment. There is an urgent need to identify biomarkers to tackle the disease's complexity and help in early diagnosis, prognosis, and therapy. Extracellular vesicles (EVs) are nanostructures released by any cell type into body fluids. Their biophysical and biochemical characteristics vary with the parent cell's physiological and pathological state and make them an attractive source of multidimensional data for patient classification and stratification. METHODS: We analyzed plasma-derived EVs of ALS patients (n = 106) and controls (n = 96), and SOD1G93A and TDP-43Q331K mouse models of ALS. We purified plasma EVs by nickel-based isolation, characterized their EV size distribution and morphology respectively by nanotracking analysis and transmission electron microscopy, and analyzed EV markers and protein cargos by Western blot and proteomics. We used machine learning techniques to predict diagnosis and prognosis. RESULTS: Our procedure resulted in high-yield isolation of intact and polydisperse plasma EVs, with minimal lipoprotein contamination. EVs in the plasma of ALS patients and the two mouse models of ALS had a distinctive size distribution and lower HSP90 levels compared to the controls. In terms of disease progression, the levels of cyclophilin A with the EV size distribution distinguished fast and slow disease progressors, a possibly new means for patient stratification. Immuno-electron microscopy also suggested that phosphorylated TDP-43 is not an intravesicular cargo of plasma-derived EVs. CONCLUSIONS: Our analysis unmasked features in plasma EVs of ALS patients with potential straightforward clinical application. We conceived an innovative mathematical model based on machine learning which, by integrating EV size distribution data with protein cargoes, gave very high prediction rates for disease diagnosis and prognosis.

Unveiling mutational dynamics in non-small cell lung cancer patients by quantitative EGFR profiling in vesicular RNA.

The mutational status of the epidermal growth factor receptor (EGFR) guides the stratification of non-small cell lung cancer (NSCLC) patients for treatment with tyrosine kinase inhibitors (TKIs). A liquid biopsy test on cell-free DNA is recommended as a clinical decision-supporting tool, although it has limited sensitivity. Here, we comparatively investigated the extracellular vesicle (EV)-RNA as an independent source for multidimensional and longitudinal EGFR profiling in a cohort of 27 NSCLC patients. We introduced and validated a new rapid, highly specific EV-RNA test with wild-type (WT) and mutant-sensitive probes (E746-A750del, L858R, and T790M). We included a cohort of 20 NSCLC patients with EGFR WT tumor tissues and systematically performed molecular EV-RNA and circulating tumor DNA analyses with clinical data statistics and biophysical profiles of EVs. At the single-patient level, we detected variegated tumor heterogeneity dynamics supported by combinations of driver EGFR mutations. EV-RNA-based mutation analysis showed an unprecedented sensitivity of over 90%. The resistance-associated mutation T790M frequently pre-existed at baseline with a gained EV-transcript copy number at progression, while the general mutational burden was mostly decreasing during the intermediate follow-up. The biophysical profile of EVs and the quantitative assessment of T790M revealed an association with tumor size determined by the sum of the longest diameters in target lesions. Vesicular RNA provides a validated tool suitable for use in clinical practice to investigate the dynamics of common driver EGFR mutations in NSCLC patients receiving TKIs.

Enzymatically active apurinic/apyrimidinic endodeoxyribonuclease 1 is released by mammalian cells through exosomes.

The apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1), the main AP-endonuclease of the DNA base excision repair pathway, is a key molecule of interest to researchers due to its unsuspected roles in different nonrepair activities, such as: i) adaptive cell response to genotoxic stress, ii) regulation of gene expression, and iii) processing of microRNAs, which make it an excellent drug target for cancer treatment. We and others recently demonstrated that APE1 can be secreted in the extracellular environment and that serum APE1 may represent a novel prognostic biomarker in hepatocellular and non-small-cell lung cancers. However, the mechanism by which APE1 is released extracellularly was not described before. Here, using three different approaches for exosomes isolation: commercial kit, nickel-based isolation, and ultracentrifugation methods and various mammalian cell lines, we elucidated the mechanisms responsible for APE1 secretion. We demonstrated that APE1 p37 and p33 forms are actively secreted through extracellular vesicles (EVs), including exosomes from different mammalian cell lines. We then observed that APE1 p33 form is generated by proteasomal-mediated degradation and is enzymatically active in EVs. Finally, we revealed that the p33 form of APE1 accumulates in EVs upon genotoxic treatment by cisplatin and doxorubicin, compounds commonly found in chemotherapy pharmacological treatments. Taken together, these findings provide for the first time evidence that a functional Base Excision Repair protein is delivered through exosomes in response to genotoxic stresses, shedding new light into the complex noncanonical biological functions of APE1 and opening new intriguing perspectives on its role in cancer biology.

Identification of a BAZ2A Bromodomain Hit Compound by Fragment Joining.

The bromodomains of BAZ2A and BAZ2B (bromodomain adjacent to zinc finger domain proteins 2) are among the most hard to drug of the 61 human bromodomains. While little is known about the role of BAZ2B, there is strong evidence for the opportunity of targeting BAZ2A in various cancers. Here, a benzimidazole-triazole fragment that binds to the BAZ2A acetyl lysine pocket was identified by a molecular docking campaign and validated by competitive binding assays and X-ray crystallography. Another ligand was observed in close proximity by soaking experiments using the BAZ2A bromodomain preincubated with the benzimidazole-triazole fragment. The crystal structure of BAZ2A with the two ligands was employed to design a few benzimidazole-triazole derivatives with increased affinity. We also present the engineering of a BAZ2A bromodomain mutant for consistent, high-resolution crystallographic studies.

Rapid Nickel-based Isolation of Extracellular Vesicles from Different Biological Fluids.

Extracellular vesicles (EVs) are membranous structures that cells massively release in extracellular fluids. EVs are cargo of cellular components such as lipids, proteins, and nucleic acids that can work as a formidable source in liquid biopsy studies searching for disease biomarkers. We recently demonstrated that nickel-based isolation (NBI) is a valuable method for fast, efficient, and easy recovery of heterogeneous EVs from biological fluids. NBI exploits nickel cations to capture negatively charged vesicles. Then, a mix of balanced chelating agents elutes EVs while preserving their integrity and stability in solution. Here, we describe steps and quality controls to functionalize a matrix of agarose beads, obtain an efficient elution of EVs, and extract nucleic acids carried by them. We demonstrate the versatility of NBI method in isolating EVs from media of primary mouse astrocytes, from human blood, urine, and saliva processed in parallel, as well as outer membrane vesicles (OMVs) from cultured Gram-negative bacteria.

RNA packaging into extracellular vesicles: An orchestra of RNA-binding proteins?

Extracellular vesicles (EVs) are heterogeneous membranous particles released from the cells through different biogenetic and secretory mechanisms. We now conceive EVs as shuttles mediating cellular communication, carrying a variety of molecules resulting from intracellular homeostatic mechanisms. The RNA is a widely detected cargo and, impressively, a recognized functional intermediate that elects EVs as modulators of cancer cell phenotypes, determinants of disease spreading, cell surrogates in regenerative medicine, and a source for non-invasive molecular diagnostics. The mechanistic elucidation of the intracellular events responsible for the engagement of RNA into EVs will significantly improve the comprehension and possibly the prediction of EV "quality" in association with cell physiology. Interestingly, the application of multidisciplinary approaches, including biochemical as well as cell-based and computational strategies, is increasingly revealing an active RNA-packaging process implicating RNA-binding proteins (RBPs) in the sorting of coding and non-coding RNAs. In this review, we provide a comprehensive view of RBPs recently emerging as part of the EV biology, considering the scenarios where: (i) individual RBPs were detected in EVs along with their RNA substrates, (ii) RBPs were detected in EVs with inferred RNA targets, and (iii) EV-transcripts were found to harbour sequence motifs mirroring the activity of RBPs. Proteins so far identified are members of the hnRNP family (hnRNPA2B1, hnRNPC1, hnRNPG, hnRNPH1, hnRNPK, and hnRNPQ), as well as YBX1, HuR, AGO2, IGF2BP1, MEX3C, ANXA2, ALIX, NCL, FUS, TDP-43, MVP, LIN28, SRP9/14, QKI, and TERT. We describe the RBPs based on protein domain features, current knowledge on the association with human diseases, recognition of RNA consensus motifs, and the need to clarify the functional significance in different cellular contexts. We also summarize data on previously identified RBP inhibitor small molecules that could also be introduced in EV research as potential modulators of vesicular RNA sorting.

Tristetraprolin/ZFP36 Regulates the Turnover of Autoimmune-Associated HLA-DQ mRNAs.

HLA class II genes encode highly polymorphic heterodimeric proteins functioning to present antigens to T cells and stimulate a specific immune response. Many HLA genes are strongly associated with autoimmune diseases as they stimulate self-antigen specific CD4+ T cells driving pathogenic responses against host tissues or organs. High expression of HLA class II risk genes is associated with autoimmune diseases, influencing the strength of the CD4+ T-mediated autoimmune response. The expression of HLA class II genes is regulated at both transcriptional and post-transcriptional levels. Protein components of the RNP complex binding the 3'UTR and affecting mRNA processing have previously been identified. Following on from this, the regulation of HLA-DQ2.5 risk genes, the main susceptibility genetic factor for celiac disease (CD), was investigated. The DQ2.5 molecule, encoded by HLA-DQA1*05 and HLA-DQB1*02 alleles, presents the antigenic gluten peptides to CD4+ T lymphocytes, activating the autoimmune response. The zinc-finger protein Tristetraprolin (TTP) or ZFP36 was identified to be a component of the RNP complex and has been described as a factor modulating mRNA stability. The 3'UTR of CD-associated HLA-DQA1*05 and HLA-DQB1*02 mRNAs do not contain canonical TTP binding consensus sequences, therefore an in silico approach focusing on mRNA secondary structure accessibility and stability was undertaken. Key structural differences specific to the CD-associated mRNAs were uncovered, allowing them to strongly interact with TTP through their 3'UTR, conferring a rapid turnover, in contrast to lower affinity binding to HLA non-CD associated mRNA.