FA-SAT ncRNA interacts with PKM2 protein: depletion of this complex induces a switch from cell proliferation to apoptosis.

FA-SAT is a highly conserved satellite DNA sequence transcribed in many Bilateria species. To disclose the cellular and functional profile of FA-SAT non-coding RNAs, a comprehensive experimental approach, including the transcripts location in the cell and in the cell cycle, the identification of its putative protein interactors, and silencing/ectopic expression phenotype analysis, was performed. FA-SAT non-coding RNAs play a nuclear function at the G1 phase of the cell cycle and the interactomic assay showed that the PKM2 protein is the main interactor. The disruption of the FA-SAT non-coding RNA/PKM2 protein complex, by the depletion of either FA-SAT or PKM2, results in the same phenotype-apoptosis, and the ectopic overexpression of FA-SAT did not affect the cell-cycle progression, but promotes the PKM2 nuclear accumulation. Overall, our data first describe the importance of this ribonucleoprotein complex in apoptosis and cell-cycle progression, what foresees a promising novel candidate molecular target for cancer therapy and diagnosis.

Future Perspectives in Detecting EGFR and ALK Gene Alterations in Liquid Biopsies of Patients with NSCLC.

Non-small-cell lung cancer (NSCLC) is a major cause of death worldwide. Alterations in such genes as EGFR and ALK are considered important biomarkers in NSCLC due to the existence of targeted therapies with specific tyrosine kinase inhibitors (TKIs). However, specific resistance-related mutations can occur during TKI treatment, which often result in therapy inefficacy. Liquid biopsies arise as a reliable tool for the early detection of these types of alterations, allowing a non-invasive follow-up of the patients. Furthermore, they can be essential for cancer screening, initial diagnosis and to check surgery success. Despite the great advantages of liquid biopsies in NSCLC and the high input that next-generation sequencing (NGS) approaches can provide in this field, its use in oncology is still limited. With improvement of assay sensitivity and the establishment of clinical guidelines for liquid biopsy analysis, it is expected that they will be used in routine procedures. This review focuses on the usefulness of liquid biopsies of NSCLC patients as a means to detect alterations in EGFR and ALK genes and in disease management, highlighting the impact of NGS methods.

Comparative Chromosome Painting in Genets (Carnivora, Viverridae, Genetta), the Only Known Feliforms with a Highly Rearranged Karyotype.

Mammalian carnivores have been extensively studied by cross-species chromosome painting, which indicated a high degree of karyotypic conservatism in the cat-like suborder Feliformia relative to the ancestral carnivore karyotype (ACK). The first exception to this high degree of karyotypic conservation in feliforms was recently confirmed in genets, mesocarnivores belonging to the basal family Viverridae. Here, we present a comparative analysis of the chromosome rearrangements among 2 subspecies of the small-spotted genet Genetta genetta (the Iberian nominate and the Arabian grantii) and the panther genet G. maculata, the 2 most common and widespread genets, using whole-chromosome paints from the domestic cat (Felis catus). The chromosome homology maps and the presence of numerous interstitial telomeric sites in both genet species strengthen the hypothesis that a highly rearranged karyotype compared to the ACK may occur throughout Genetta. The karyotype of G. maculata appears to have undergone more rearrangements than that of G. genetta, which is an older lineage. Notably, we identified a tandem fusion distinguishing G. g. genetta and G. g.grantii. As G. g. grantii is morphologically and genetically distinctive, and tandem fusions have been associated with substantial postzygotic isolation in mammals, this cytogenetic finding flags the subspecies for future taxonomic investigations.

Classical and Molecular Cytogenetics of the Panther Genet Genetta maculata (Mammalia, Carnivora, Viverridae).

Genets (Genetta) are a genus of African mammalian carnivorans with 14 currently recognized species, although taxonomic uncertainties remain, particularly regarding the number of species within the large-spotted genet complex. This study presents the first banded karyotype and molecular cytogenetic analysis of a genetically identified panther genet, Genetta maculata, the most common and widespread taxon of the large-spotted genet complex, with a wide distribution in sub-Saharan Africa. Sampled in Gauteng Province, South Africa, it could be assigned to the subspecies G. m. letabae on geographic grounds and had a similar karyotype (2n = 52, FNa = 96) to those published for a panther genet from Ethiopia and for the West African large-spotted genet G. pardina. Notably, the specimen had a different autosomal morphology (2 acrocentric chromosomes) from that previously attributed to letabae (a single acrocentric chromosome), but the latter assignment was uncertain because the studied individuals were captive born and assigned based solely on a presumed origin in the former Transvaal Province of South Africa. Fluorescence in situ hybridization with a telomere repeat probe revealed the presence of telomeric sequences in the centromeres of most chromosomes, the so-called interstitial telomeric sites (ITSs). Since genets seem to have a unique, highly rearranged karyotype among feliforms and relatively low interspecific karyotypic variation, and considering the known instability of ITSs, we suggest that the large amount of ITSs found here might be due to evolutionarily recent extensive genomic rearrangements. This study provides cytogenetic information that contributes to our understanding of chromosomal variation and genomic rearrangements in genets, and valuable baseline data for future studies of karyotype evolution in carnivores in general and viverrids in particular.

The puzzling character of repetitive DNA in Phodopus genomes (Cricetidae, Rodentia).

Three novel repetitive DNA sequences are described, presenting a similar heterochromatic chromosomal location in two hamster species: Phodopus roborovskii and Phodopus sungorus (Cricetidae, Rodentia). Namely, two species-specific repetitive sequences (PROsat from P. roborovskii and PSUchr1sat from P. sungorus) surrounding a third one (PsatDNA), that is shared by both hamster genomes. Fiber-FISH analyses revealed that PROsat intermingles with PsatDNA in P. roborovskii and PSUchr1sat intermingles with PsatDNA in P. sungorus. A model explaining the evolution of this intricate chromosomal distribution is proposed, which can explain better the evolution of these very derivative genomes (in comparison to the ancestral Muroidea). The most plausible evolutionary scenario seems to be the expansion of a number of repeats into other's domain, most probably resulting in its intermingling, followed by the subsequent spread of these complex repeats from a single chromosomal location to other chromosomes. Evidences of an association between repetitive sequences and the chromosome evolution process were observed, namely for PROsat. Most probably, the evolutionary breakpoints that shaped PRO and PSU chromosomes (pericentric inversions and fusions) occurred within the boundaries of PROsat blocks in the ancestor. The repeats high diversity at the heterochromatic regions of Phodopus chromosomes, together with its complex organization, suggests that these species are important models for evolutionary studies, namely in the investigation of a possible relationship between repetitive sequences and the occurrence of chromosomal rearrangements and consequently, in genome evolution.

Satellite non-coding RNAs: the emerging players in cells, cellular pathways and cancer.

For several decades, transcriptional inactivity was considered as one of the particular features of constitutive heterochromatin and, therefore, of its major component, satellite DNA sequences. However, more recently, succeeding evidences have demonstrated that these sequences can indeed be transcribed, yielding satellite non-coding RNAs with important roles in the organization and regulation of genomes. Since then, several studies have been conducted, trying to understand the function(s) of these sequences not only in the normal but also in cancer genomes. It is thought that the association between cancer and satncRNAs is mostly due to the influence of these transcripts in the genome instability, a hallmark of cancer. The few reports on satellite DNA transcription in cancer contexts point to its overexpression; however, this scenario may be far more complex, variable, and influenced by a number of factors and the exact role of satncRNAs in the oncogenic process remains poorly understood. The greater is the knowledge on the association of satncRNAs with cancer, the greater would be the opportunity to assist cancer treatment, either by the design of effective therapies targeting these molecules or by using them as biomarkers in cancer diagnosis, prognosis, and with predictive value.

Cytogenetic Assessment of the Rat Cell Line CLS-ACI-1: An in vitro Cell Model for Mycn Overexpression.

Breast cancer is a complex and heterogeneous disease, and the establishment of cell models in order to properly study the disease at the molecular and cellular level is of utmost importance. Here, we present the cytogenetic characterization and gene expression analysis of the tumoral mammary rat cell line CLS-ACI-1. The use of banding and molecular cytogenetic techniques allowed the description of the complex CLS-ACI-1 karyotype and the identification of breakpoints in clonal chromosome rearrangements. Moreover, a Mycn and Erbb2 comparative expression analysis by RT-qPCR was performed, revealing a high expression level of Mycn in CLS-ACI-1 cells. Moreover, a considerable number of putative mutated genes and chromosome alterations detected through cytogenetic analysis seem to be in the MYCN biological network. Therefore, the CLS-ACI-1 cell line is presented as a promising cell model for the study of the role of MYCN in breast cancer and also as a tool for developing appropriate cancer therapies, namely for Mycn targeting.

A High-Resolution Comparative Chromosome Map of Cricetus cricetus and Peromyscus eremicus Reveals the Involvement of Constitutive Heterochromatin in Breakpoint Regions.

Compared to humans and other mammals, rodent genomes, specifically Muroidea species, underwent intense chromosome reshuffling in which many complex structural rearrangements occurred. This fact makes them preferential animal models for studying the process of karyotype evolution. Here, we present the first combined chromosome comparative maps between 2 Cricetidae species, Cricetus cricetus and Peromyscus eremicus, and the index species Mus musculus and Rattus norvegicus. Comparative chromosome painting was done using mouse and rat paint probes together with in silico analysis from the Ensembl genome browser database. Hereby, evolutionary events (inter- and intrachromosomal rearrangements) that occurred in C. cricetus and P. eremicus since the putative ancestral Muroidea genome could be inferred, and evolutionary breakpoint regions could be detected. A colocalization of constitutive heterochromatin and evolutionary breakpoint regions in each genome was observed. Our results suggest the involvement of constitutive heterochromatin in karyotype restructuring of these species, despite the different levels of conservation of the C. cricetus (derivative) and P. eremicus (conserved) genomes.

A novel satellite DNA sequence in the Peromyscus genome (PMSat): Evolution via copy number fluctuation.

Satellite DNAs (satDNA) are tandemly arrayed repeated sequences largely present in eukaryotic genomes, which play important roles in genome evolution and function, and therefore, their analysis is vital. Here, we describe the isolation of a novel satellite DNA family (PMSat) from the rodent Peromyscus eremicus (Cricetidae, Rodentia), which is located in pericentromeric regions and exhibits a typical satellite DNA genome organization. Orthologous PMSat sequences were isolated and characterized from three species belonging to Cricetidae: Cricetus cricetus, Phodopus sungorus and Microtus arvalis. In these species, PMSat is highly conserved, with the absence of fixed species-specific mutations. Strikingly, different numbers of copies of this sequence were found among the species, suggesting evolution by copy number fluctuation. Repeat units of PMSat were also found in the Peromyscus maniculatus bairdii BioProject, but our results suggest that these repeat units are from genome regions outside the pericentromere. The remarkably high evolutionary sequence conservation along with the preservation of a few numbers of copies of this sequence in the analyzed genomes may suggest functional significance but a different sequence nature/organization. Our data highlight that repeats are difficult to analyze due to the limited tools available to dissect genomes and the fact that assemblies do not cover regions of constitutive heterochromatin.

Praomys tullbergi (Muridae, Rodentia) genome architecture decoded by comparative chromosome painting with Mus and Rattus.

The order Rodentia and in particular the Muridae are characterised by extremely high rates of chromosome evolution and remarkable chromosome diversity. The Praomys group (Murinae, Muridae and Rodentia) constitutes a diverse and abundant group divided into two complexes, the jacksoni complex and the tullbergi complex which includes the species Praomys tullbergi. Comparative chromosome painting using the two index genomes, Mus musculus and Rattus norvegicus, was performed resulting in a high resolution chromosome map for P. tullbergi. The combined use of rat and mouse probes and the assistance of the assembly of all the available sequencing data from Ensembl genome browser allowed a great dissection of P. tullbergi genome, the detection of inversion events and ultimately the refinement of P. tullbergi comparative map. A key achievement was the reconstruction of a high precision Muroidea ancestral karyotype (Muridae/Cricetidae and Murine) based in a broad species analysis combining previous reported comparative maps together with the presented data. This permitted the reconstruction of the evolutionary history of chromosome changes since the ancestral Muroidea genome and enlightened the phylogenetic relationships with the related species mouse and rat. The analysis of constitutive heterochromatin and its co-localisation with the identified evolutionary breakpoints regions was performed suggesting the involvement of repetitive sequences in the chromosome rearrangements that originated the present P. tullbergi genome architecture.

pH-Responsive Hybrid Nanoassemblies for Cancer Treatment: Formulation Development, Optimization, and In Vitro Therapeutic Performance.

Current needs for increased drug delivery carrier efficacy and specificity in cancer necessitate the adoption of intelligent materials that respond to environmental stimuli. Therefore, we developed and optimized pH-triggered drug delivery nanoassemblies that exhibit an increased release of doxorubicin (DOX) in acidic conditions typical of cancer tissues and endosomal vesicles (pH 5.5) while exhibiting significantly lower release under normal physiological conditions (pH 7.5), indicating the potential to reduce cytotoxicity in healthy cells. The hybrid (polymeric/lipid) composition of the lyotropic non-lamellar liquid crystalline (LNLCs) nanoassemblies demonstrated high encapsulation efficiency of the drug (>90%) and high drug loading content (>7%) with colloidal stability lasting at least 4 weeks. Confocal microscopy revealed cancer cellular uptake and DOX-loaded LNLCs accumulation near the nucleus of human hepatocellular carcinoma cells, with a large number of cells appearing to be in apoptosis. DOX-loaded LNLCs have also shown higher citotoxicity in cancer cell lines (MDA-MB 231 and HepG2 cell lines after 24 h and in NCI-H1299 cell line after 48 h) when compared to free drug. After 24 h, free DOX was found to have higher cytotoxicity than DOX-loaded LNLCs and empty LNLCs in the normal cell line. Overall, the results demonstrate that DOX-loaded LNLCs have the potential to be explored in cancer therapy.

Development of a QCM-based biosensor for the detection of non-small cell lung cancer biomarkers in liquid biopsies.

Lung cancer is the main malignant cancer reported worldwide, with one of the lowest survival rates. Deletions in the Epidermal Growth Factor Receptor (EGFR) gene are often associated with non-small cell lung cancer (NSCLC), a common subtype of lung cancer. The detection of such mutations provides key information for the diagnosis and treatment of the disease; therefore, the early screening of such biomarkers is of vital importance. The need for fast, reliable, and early detection means applied to NSCLC has led to the development of highly sensitive devices that can detect cancer-associated mutations. Such devices, known as biosensors, are a promising alternative to more conventional detection methods and can potentially alter the way cancer is diagnosed and treated. In this study, we report the development of a DNA-based biosensor, namely a quartz crystal microbalance (QCM), applied to the detection of NSCLC, from liquid biopsies samples. The detection, as is the case of most DNA biosensors, is based on the hybridization between the NSCLC-specific probe and the sample DNA (containing specific mutations associated with NSCLC). The surface functionalization was performed with a blocking agent (dithiothreitol) and thiolated-ssDNA strands. The biosensor was able to detect specific DNA sequences in both synthetic and real samples. Aspects such as reutilization and regeneration of the QCM electrode were also studied.

Natural Compounds: Co-Delivery Strategies with Chemotherapeutic Agents or Nucleic Acids Using Lipid-Based Nanocarriers.

Cancer is one of the leading causes of death, and latest predictions indicate that cancer- related deaths will increase over the next few decades. Despite significant advances in conventional therapies, treatments remain far from ideal due to limitations such as lack of selectivity, non-specific distribution, and multidrug resistance. Current research is focusing on the development of several strategies to improve the efficiency of chemotherapeutic agents and, as a result, overcome the challenges associated with conventional therapies. In this regard, combined therapy with natural compounds and other therapeutic agents, such as chemotherapeutics or nucleic acids, has recently emerged as a new strategy for tackling the drawbacks of conventional therapies. Taking this strategy into consideration, the co-delivery of the above-mentioned agents in lipid-based nanocarriers provides some advantages by improving the potential of the therapeutic agents carried. In this review, we present an analysis of the synergistic anticancer outcomes resulting from the combination of natural compounds and chemotherapeutics or nucleic acids. We also emphasize the importance of these co-delivery strategies when reducing multidrug resistance and adverse toxic effects. Furthermore, the review delves into the challenges and opportunities surrounding the application of these co-delivery strategies towards tangible clinical translation for cancer treatment.

Chromosomal evolution in Rattini (Muridae, Rodentia).

The Rattini (Muridae, Murinae) includes the biologically important model species Rattus norvegicus (RNO) and represents a group of rodents that are of clinical, agricultural and epidemiological importance. We present a comparative molecular cytogenetic investigation of ten Rattini species representative of the genera Maxomys, Leopoldamys, Niviventer, Berylmys, Bandicota and Rattus using chromosome banding, cross-species painting (Zoo-fluorescent in situ hybridization or FISH) and BAC-FISH mapping. Our results show that these taxa are characterised by slow to moderate rates of chromosome evolution that contrasts with the extensive chromosome restructuring identified in most other murid rodents, particularly the mouse lineage. This extends to genomic features such as NOR location (for example, NORs on RNO 3 are present on the corresponding chromosomes in all species except Bandicota savilei and Niviventer fulvescens, and the NORs on RNO 10 are conserved in all Rattini with the exception of Rattus). The satellite I DNA family detected and characterised herein appears to be taxon (Rattus) specific, and of recent origin (consistent with a feedback model of satellite evolution). BAC-mapping using clones that span regions responsible for the morphological variability exhibited by RNO 1, 12 and 13 (acrocentric/submetacentric) and their orthologues in Rattus species, demonstrated that the differences are most likely due to pericentric inversions as exemplified by data on Rattus tanezumi. Chromosomal characters detected using R. norvegicus and Maxomys surifer whole chromosome painting probes were mapped to a consensus sequence-based phylogenetic tree thus allowing an objective assessment of ancestral states for the reconstruction of the putative Rattini ancestral karyotype. This is thought to have comprised 46 chromosomes that, with the exception of a single pair of metacentric autosomes, were acrocentric in morphology.

Comprehensive In Silico Analysis of Retrotransposon Insertions within the Survival Motor Neuron Genes Involved in Spinal Muscular Atrophy.

Transposable elements (TEs) are interspersed repetitive and mobile DNA sequences within the genome. Better tools for evaluating TE-derived sequences have provided insights into the contribution of TEs to human development and disease. Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease that is caused by deletions or mutations in the Survival Motor Neuron 1 (SMN1) gene but retention of its nearly perfect orthologue SMN2. Both genes are highly enriched in TEs. To establish a link between TEs and SMA, we conducted a comprehensive, in silico analysis of TE insertions within the SMN1/2 loci of SMA, carrier and healthy genomes. We found an Alu insertion in the promoter region and one L1 element in the 3'UTR that may play an important role in alternative promoter as well as in alternative transcriptional termination. Additionally, several intronic Alu repeats may influence alternative splicing via RNA circularization and causes the presence of new alternative exons. These Alu repeats present throughout the genes are also prone to recombination events that could lead to SMN1 exons deletions and, ultimately, SMA. TE characterization of the SMA genomic region could provide for a better understanding of the implications of TEs on human disease and genomic evolution.

Satellite Noncoding RNAs (ncRNA) as Cancer Biomarkers? New Insights from FA-SAT ncRNA Molecular and Clinical Profiles in Feline Mammary Tumors.

Satellite noncoding RNAs (ncRNAs) are a new frontier of cancer biology research and biomarkers. While the knowledge on ncRNAs in human cancers is still limited, studies in other species can be informative to guide future translational research and development for cancer molecular targets and diagnostics. In this context, FA-SAT is the major satellite DNA of the cat genome, which is also present in humans, being transcribed in both species. In this study, we report new insights on FA-SAT (DNA and RNA) profile in feline mammary tumors, using disease-free tissues from the same animals as reference. We quantified the FA-SAT DNA and RNA levels (long and small transcripts) by real-time quantitative polymerase chain reaction (qPCR) and RT-qPCR. The comparison of the FA-SAT DNA and RNA levels with clinicopathological parameters revealed several associations, such as (1) the FA-SAT DNA levels' positive relation with lymphovascular invasion, (2) the FA-SAT long RNA negative correlation with Ki-67 index, and its positive association with Estrogen Receptor status, and (3) the FA-SAT small RNA level positive correlation with tumor size and skin ulceration. Also, FA-SAT long RNA is correlated with ERBB2 and c-MYC RNA levels. These data collectively suggest that FA-SAT ncRNA offers prospects as a potential cancer biomarker in cats. Further studies in humans are also needed to decipher the emerging role of ncRNAs in cancer biology and precision medicine fields. This work brings new information on the relation of FA-SAT ncRNAs with the oncogenic process, uncovering a new potential cancer biomarker.

Decoding the Role of Satellite DNA in Genome Architecture and Plasticity-An Evolutionary and Clinical Affair.

Repetitive DNA is a major organizational component of eukaryotic genomes, being intrinsically related with their architecture and evolution. Tandemly repeated satellite DNAs (satDNAs) can be found clustered in specific heterochromatin-rich chromosomal regions, building vital structures like functional centromeres and also dispersed within euchromatin. Interestingly, despite their association to critical chromosomal structures, satDNAs are widely variable among species due to their high turnover rates. This dynamic behavior has been associated with genome plasticity and chromosome rearrangements, leading to the reshaping of genomes. Here we present the current knowledge regarding satDNAs in the light of new genomic technologies, and the challenges in the study of these sequences. Furthermore, we discuss how these sequences, together with other repeats, influence genome architecture, impacting its evolution and association with disease.

Hidden heterochromatin: Characterization in the Rodentia species Cricetus cricetus, Peromyscus eremicus (Cricetidae) and Praomys tullbergi (Muridae).

The use of in situ restriction endonuclease (RE) (which cleaves DNA at specific sequences) digestion has proven to be a useful technique in improving the dissection of constitutive heterochromatin (CH), and in the understanding of the CH evolution in different genomes. In the present work we describe in detail the CH of the three Rodentia species, Cricetus cricetus, Peromyscus eremicus (family Cricetidae) and Praomys tullbergi (family Muridae) using a panel of seven REs followed by C-banding. Comparison of the amount, distribution and molecular nature of C-positive heterochromatin revealed molecular heterogeneity in the heterochromatin of the three species. The large number of subclasses of CH identified in Praomys tullbergi chromosomes indicated that the karyotype of this species is the more derived when compared with the other two genomes analyzed, probably originated by a great number of complex chromosomal rearrangements. The high level of sequence heterogeneity identified in the CH of the three genomes suggests the coexistence of different satellite DNA families, or variants of these families in these genomes.

DNA Methylation Patterns of a Satellite Non-coding Sequence - FA-SAT in Cancer Cells: Its Expression Cannot Be Explained Solely by DNA Methylation.

Satellite ncRNAs are emerging as key players in cell and cancer pathways. Cancer-linked satellite DNA hypomethylation seems to be responsible for the overexpression of satellite non-coding DNAs in several tumors. FA-SAT is the major satellite DNA of Felis catus and recently, its presence and transcription was described across Bilateria genomes. This satellite DNA is GC-rich and includes a CpG island, what is suggestive of transcription regulation via DNA methylation. In this work, it was studied for the first time the FA-SAT methylation profile in cat primary cells, in four passages of the cat tumor cell line FkMTp and in eight feline mammary tumors and the respective disease-free tissues. Contrary to what was expected, we found that in most of the tumor samples analyzed, FA-SAT DNA was not hypomethylated. Furthermore, in these samples the transcription of FA-SAT does not correlate with the methylation status. The use of a global demethylating agent, 5-Azacytidine, in cat primary cells caused an increase in the FA-SAT non-coding RNA levels. However, global demethylation in the tumor FkMTp cells only resulted in the increased levels of the FA-SAT small RNA fraction. Our data suggests that DNA methylation of FA-SAT is involved in the regulation of this satellite DNA, however, other mechanisms are certainly contributing to the transcriptional status of the sequence, specifically in cancer.

Gene expression association study in feline mammary carcinomas.

Works on cancer-related genes expression using feline mammary carcinomas (FMCs) are scarce but crucial, not only to validate these tumours as models for human breast cancer studies but also to improve small animal practice. Here, the expression of the cancer-related genes TP53, CCND1, FUS, YBX1, PTBP1, c-MYC and PKM2 was evaluated by real-time RT-qPCR, in a population of FMCs clinically characterized and compared with the disease-free tissue of the same individual. In most of the FMCs analysed, RNA quantification revealed normal expression levels for TP53, c-MYC, YBX1 and FUS, but overexpression in the genes CCND1, PTBP1 and PKM2. The expression levels of these cancer-related genes are strongly correlated with each other, with exception of c-MYC and PKM2 genes. The integration of clinicopathological data with the transcriptional levels revealed several associations. The oral contraceptive administration showed to be positively related with the TP53, YBX1, CCND1, FUS and PTBP1 RNA levels. Positive associations were found between tumour size and YBX1 RNA, and lymph node metastasis with c-MYC RNA levels. This work allowed to verify that many of these cancer-related genes are associated but may also, indirectly, influence other genes, creating a complex molecular cancer network that in the future can provide new cancer biomarkers.