Epithelial-mesenchymal transition associated markers in sarcomatoid transformation of clear cell renal cell carcinoma.

Epithelial-mesenchymal transition (EMT) plays a pivotal role in the development and progression of many cancers. Partial EMT (pEMT) could represent a critical step in tumor migration and dissemination. Sarcomatoid renal cell carcinoma (sRCC) is an aggressive form of renal cell carcinoma (RCC) composed of a carcinomatous (sRCC-Ca) and sarcomatous (sRCC-Sa) component. The role of (p)EMT in the progression of RCC to sRCC remains unclear. The aim of this study was to investigate the involvement of (p)EMT in RCC and sRCC. Tissue samples from 10 patients with clear cell RCC (ccRCC) and 10 patients with sRCC were selected. The expression of main EMT markers (miR-200 family, miR-205, SNAI1/2, TWIST1/2, ZEB1/2, CDH1/2, VIM) was analyzed by qPCR in ccRCC, sRCC-Ca, and sRCC-Sa and compared to non-neoplastic tissue and between both groups. Expression of E-cadherin, N-cadherin, vimentin and ZEB2 was analyzed using immunohistochemistry. miR-200c was downregulated in sRCC-Ca compared to ccRCC, while miR-200a was downregulated in sRCC-Sa compared to ccRCC. CDH1 was downregulated in sRCC-Sa when compared to any other group. ZEB2 was downregulated in ccRCC and sRCC compared to corresponding non-neoplastic kidney. A positive correlation was observed between CDH1 expression and miR-200a/b/c. Our results suggest that full EMT is not present in sRCC. Instead, discreet molecular differences exist between ccRCC, sRCC-Ca, and sRCC-Sa, possibly representing distinct intermediary states undergoing pEMT.

Circulating microRNA Panels for Detection of Liver Cancers and Liver-Metastasizing Primary Cancers.

Malignant liver tumors, including primary malignant liver tumors and liver metastases, are among the most frequent malignancies worldwide. The disease carries a poor prognosis and poor overall survival, particularly in cases involving liver metastases. Consequently, the early detection and precise differentiation of malignant liver tumors are of paramount importance for making informed decisions regarding patient treatment. Significant research efforts are currently directed towards the development of diagnostic tools for different types of cancer using minimally invasive techniques. A prominent area of focus within this research is the evaluation of circulating microRNA, for which dysregulated expression is well documented in different cancers. Combining microRNAs in panels using serum or plasma samples derived from blood holds great promise for better sensitivity and specificity for detection of certain types of cancer.

In Silico Gene Prioritization Highlights the Significance of Bone Morphogenetic Protein 4 (BMP4) Promoter Methylation across All Methylation Clusters in Colorectal Cancer.

The cytosine-phosphate-guanine (CpG) island methylator phenotype (CIMP) represents one of the pathways involved in the development of colorectal cancer, characterized by genome-wide hypermethylation. To identify samples exhibiting hypermethylation, we used unsupervised hierarchical clustering on genome-wide methylation data. This clustering analysis revealed the presence of four distinct subtypes within the tumor samples, namely, CIMP-H, CIMP-L, cluster 3, and cluster 4. These subtypes demonstrated varying levels of methylation, categorized as high, intermediate, and very low. To gain further insights, we mapped significant probes from all clusters to Ensembl Regulatory build 89, with a specific focus on those located within promoter regions or bound regions. By intersecting the methylated promoter and bound regions across all methylation subtypes, we identified a total of 253 genes exhibiting aberrant methylation patterns in the promoter regions across all four subtypes of colorectal cancer. Among these genes, our comprehensive genome-wide analysis highlights bone morphogenic protein 4 (BMP4) as the most prominent candidate. This significant finding was derived through the utilization of various bioinformatics tools, emphasizing the potential role of BMP4 in colorectal cancer development and progression.

Association between histopathological changes and expression of selected microRNAs in skin of adult patients with IgA vasculitis.

AIMS: IgA vasculitis (IgAV) is a common small-vessel systemic vasculitisthat is histologically characterised by granulocyte infiltration and IgA deposition in vessel walls. Information on microRNA (miRNA) involvement inIgAVis limited. The aim of this study was to analyse the association between histopathological changes and expression profiles of 14 miRNAs in the affected skin of 70 adult patients with IgAV. METHODS AND RESULTS: miRNA expression analysis was performed by quantitative real-time polymerase chain reaction and evaluation of histopathological changes by light and immunofluorescence microscopy on formalin-fixed paraffin-embedded skin excision samples. In IgAV-affected skin, granulocyte infiltration was significantly associated with vessel fibrinoid necrosis. Of the analysed miRNAs, four showed two-fold increased expression (let-7d, let-7f, miR-21-5p, and miR-203-3p), five showed five-fold increased expression (let-7b, miR-17-5p, miR-155-5p, miR-423-5p, and miR-451a), and threeshowed 15-fold increased expression (let-7a, miR-21-3p, miR-223-3p), as compared with controls (all P < 0.001). miR-146a-5p and miR-148b-3p showed three-fold decreased expression (P = 0.981 and P < 0.001). The expression of miR-223-3p also showed a significant positive association with granulocyte infiltration and fibrinoid necrosis. CONCLUSIONS: Altered miRNA expression, especially of miRNA-223-3p, may be associated with the skin inflammatory state in IgAV. The majority of aberrantly expressed miRNAs in IgAV-affected skin are known to influence the nuclear factor-κB signalling pathway, which is crucial for activation of key proinflammatory genes, including those encoding tumour necrosis factor-α, interleukin (IL)-6, and IL-8. Furthermore, miR-146a-5p and miR-148b-3p, which are negative regulators of inflammatory gene expression, showed decreased expression and could contribute to the exaggerated inflammation. Further investigation of miRNA expression in the affected tissues could improve our knowledge of IgAV pathogenesis, and possibly help to identify novel biomarkers in body fluids.

The maternal perspective for five Slovenian regions: The importance of regional sampling.

BACKGROUND: The Slovenian territory is geographically positioned between the Alps, Adriatic Sea, Pannonian basin and the Dinaric Mountains and, as such, has served as a passageway for various populations in different periods of time. Turbulent historic events and diverse geography of the region have produced a diverse contemporary population whose genetic analysis could provide insight into past demographic events. AIM: The aims of this study were to characterize the Slovenian mitochondrial gene pool at the micro-geographic level and to compare it with surrounding populations. SUBJECTS AND METHODS: A total of 402 individuals from five Slovenian regions were analysed in this study by typing HVR I, HVR II and coding region polymorphisms of mtDNA. RESULTS: Analysis revealed 47 haplogroups and sub-haplogroups, the most common of which were H*, H1, J1c, T2 and U5a. Intra-population comparisons revealed a sharp gradient of the J1c haplogroup between Slovenian regions, with a peak frequency of 24.5% being observed in the population of the Littoral Region. CONCLUSION: The sharp gradient of the J1c haplogroup between Slovenian regions is in line with the archaeological horizon known as Impressed Ware culture and could, therefore, represent a genetic trace of the early Neolithic expansion route along the East Adriatic coastal region.

Discovery of novel DNA methylation biomarker panels for the diagnosis and differentiation between common adenocarcinomas and their liver metastases.

Differentiation between adenocarcinomas is sometimes challenging. The promising avenue for discovering new biomarkers lies in bioinformatics using DNA methylation analysis. Utilizing a 2853-sample identification dataset and a 782-sample independent verification dataset, we have identified diagnostic DNA methylation biomarkers that are hypermethylated in cancer and differentiate between breast invasive carcinoma, cholangiocarcinoma, colorectal cancer, hepatocellular carcinoma, lung adenocarcinoma, pancreatic adenocarcinoma and stomach adenocarcinoma. The best panels for cancer type exhibit sensitivity of 77.8-95.9%, a specificity of 92.7-97.5% for tumors, a specificity of 91.5-97.7% for tumors and normal tissues and a diagnostic accuracy of 85.3-96.4%. We have shown that the results can be extended from the primary cancers to their liver metastases, as the best panels diagnose and differentiate between pancreatic adenocarcinoma liver metastases and breast invasive carcinoma liver metastases with a sensitivity and specificity of 83.3-100% and a diagnostic accuracy of 86.8-91.9%. Moreover, the panels could detect hypermethylation of selected regions in the cell-free DNA of patients with liver metastases. At the same time, these were unmethylated in the cell-free DNA of healthy donors, confirming their applicability for liquid biopsies.

AmiCa: Atlas of miRNA-gene correlations in cancer.

The increasing availability of RNA sequencing data has opened up numerous opportunities to analyze various RNA interactions, including microRNA-target interactions (MTIs). In response to the necessity for a specialized tool to study MTIs in cancer and normal tissues, we developed AmiCa (https://amica.omics.si/), a web server designed for comprehensive analysis of mature microRNA (miRNA) and gene expression in 32 cancer types. Data from 9498 tumor samples and 626 normal samples from The Cancer Genome Atlas were obtained through the Genomic Data Commons and used to calculate differential expression and miRNA-target gene (MTI) correlations. AmiCa provides data on differential expression of miRNAs/genes for cancers for which normal tissue samples were available. In addition, the server calculates and presents correlations separately for tumor and normal samples for cancers for which normal samples are available. Furthermore, it enables the exploration of miRNA/gene expression in all cancer types with different miRNA/gene expression. In addition, AmiCa includes a ranking system for genes and miRNAs that can be used to identify those that are particularly highly expressed in certain cancers compared to other cancers, facilitating targeted and cancer-specific research. Finally, the functionality of AmiCa is illustrated by two case studies.

Preservation of 5-Hydroxymethylcytosine Levels in LRIG1 across Genomic DNA and Cell-Free DNA in Glioma Patients.

Cell-free DNA (cfDNA) has recently emerged as a promising minimally invasive diagnostic biomarker for various cancers. In this study, our aim was to identify cfDNA biomarkers by investigating genes that displayed significant differences between glioma patients and their corresponding controls. To accomplish this, we utilized publicly available data from the Gene Expression Omnibus, focusing on 5-hydroxymethylcytosine (5hmC) profiles in both cfDNA and genomic DNA (gDNA) from glioma patients and healthy individuals. The intersection of gene lists derived from these comparative analyses unveiled LRIG1 and ZNF703 as the two genes with elevated 5hmC levels in both the cfDNA of glioma patients and gDNA of glioma tissue compared to their respective controls. The gene expression data revealed both genes were upregulated in glioma tissue compared to normal brain tissue. Integration of 5hmC data revealed a strong positive correlation in the glioma tissue group between 5hmC and the gene expression of the LRIG1 gene. Furthermore, exploration using the AmiCa web tool indicated that LRIG1 gene expression was elevated compared to 17 other cancers included in the database, emphasizing its potential as a distinctive biomarker across multiple cancer types.

Thrombospondin 2, matrix Gla protein and digital analysis identified distinct fibroblast populations in fibrostenosing Crohn's disease.

Fibrosis is an important complication in inflammatory bowel diseases. Previous studies suggest an important role of matrix Gla protein (MGP) and thrombospondin 2 (THBS2) in fibrosis in various organs. Our aim was to analyse their expression together with regulatory miRNAs in submucosal and subserosal fibroblasts in ulcerative colitis (UC) and Crohn's disease (CD) using immunohistochemistry and qPCR. Digital pathology was used to compare collagen fibre characteristics of submucosal and subserosal fibrosis. Immunohistochemistry showed expression of MGP, but not THBS2 in submucosa in UC and CD. In the subserosa, there was strong staining for both proteins in CD but not in UC. qPCR showed significant upregulation of THBS2 and MGP genes in CD subserosa compared to the submucosa. Digital pathology analysis revealed higher proportion of larger and thicker fibres that were more tortuous and reticulated in subserosal fibrosis compared to submucosal fibrosis. These results suggest distinct fibroblast populations in fibrostenosing CD, and are further supported by image analysis showing significant differences in the morphology and architecture of collagen fibres in submucosal fibrosis in comparison to subserosal fibrosis. Our study is the first to describe differences in submucosal and subserosal fibroblast populations, contributing to understanding of the pathogenesis of fibrostenosis in CD.

Long non-coding RNA in cancer.

Long non-coding RNAs (lncRNAs) are pervasively transcribed in the genome and are emerging as new players in tumorigenesis due to their various functions in transcriptional, posttranscriptional and epigenetic mechanisms of gene regulation. LncRNAs are deregulated in a number of cancers, demonstrating both oncogenic and tumor suppressive roles, thus suggesting their aberrant expression may be a substantial contributor in cancer development. In this review, we will summarize their emerging role in human cancer and discuss their perspectives in diagnostics as potential biomarkers.

MicroRNAs and long non-coding RNAs: prospects in diagnostics and therapy of cancer.

BACKGROUND: Non-coding RNAs (ncRNAs) are key regulatory molecules in cellular processes, and are potential biomarkers in many diseases. Currently, microRNAs and long non-coding RNAs are being pursued as diagnostic and prognostic biomarkers, and as therapeutic tools in cancer, since their expression profiling is able to distinguish different cancer types and classify their sub-types. CONCLUSIONS: There are numerous studies confirming involvement of ncRNAs in cancer initiation, development and progression, but have only been recently identified as new diagnostic and prognostic tools. This can be beneficial in future medical cancer treatment options, since ncRNAs are natural antisense interactors included in regulation of many genes connected to survival and proliferation. Research is directed in development of useful markers for diagnosis and prognosis in cancer and in developing new RNA-based cancer therapies, of which some are already in clinical trials.

Circulating Tumor DNA Methylation Biomarkers for Characterization and Determination of the Cancer Origin in Malignant Liver Tumors.

Malignant liver tumors include primary malignant liver tumors and liver metastases. They are among the most common malignancies worldwide. The disease has a poor prognosis and poor overall survival, especially with liver metastases. Therefore, early detection and differentiation between malignant liver tumors are critical for patient treatment selection. The detection of cancer and the prediction of its origin is possible with a DNA methylation profile of the tumor DNA compared to that of normal cells, which reflects tissue differentiation and malignant transformation. New technologies enable the characterization of the tumor methylome in circulating tumor DNA (ctDNA), providing a variety of new ctDNA methylation biomarkers, which can provide additional information to clinical decision-making. Our review of the literature provides insight into methylation changes in ctDNA from patients with common malignant liver tumors and can serve as a starting point for further research.

Steadfast Toll Like Receptor 4 (TLR4) 5-Hydroxymethylcytosine Levels in Cell-Free DNA: A Promising Consistency Marker for Colorectal Cancer Patients.

Cell-free DNA (cfDNA) from patient blood is emerging as a noninvasive diagnostic avenue for various cancers. We aimed to identify reliable biomarkers in cfDNA by investigating genes exhibiting significant differences between colorectal cancer and control samples. Our objective was to identify genes that showed a positive difference between cancer and control samples. To achieve this, we conducted an in silico analysis to identify genes that exhibit no significant variation in methylation between genomic DNA (gDNA) and cfDNA. We collected experimental data from publicly available repositories, which included 5-hydroxymethylcytosine (5hmC) profiles of gDNA and cfDNA samples from both cancer patients and healthy individuals. By comparing and overlapping these two groups, we identified 187 genes of interest, of which 53 genes had a positive difference among colon cancer patients and healthy individuals. Next, we performed an ANOVA test on these genes, resulting in the identification of 12 genes that showed statistically significant higher levels of 5hmC in cfDNA and gDNA from cancer patients compared to healthy individuals. Additionally, we compared the 5hmC status of these genes between cfDNA and gDNA from cancer patients. Interestingly, we found that the 5hmC of the toll like receptor 4 (TLR4) gene was not statistically different between cfDNA and gDNA from cancer patients, indicating consistency between cfDNA and gDNA. These findings have important implications, not only for experimental validation but also for the development of more sensitive and robust noninvasive methods to improve diagnostic, prognostic, and treatment options for colon cancer.

Long Non-Coding RNAs as Potential Regulators of EMT-Related Transcription Factors in Colorectal Cancer-A Systematic Review and Bioinformatics Analysis.

Epithelial-mesenchymal transition (EMT) plays a pivotal role in carcinogenesis, influencing cancer progression, metastases, stemness, immune evasion, metabolic reprogramming and therapeutic resistance. EMT in most carcinomas, including colorectal carcinoma (CRC), is only partial, and can be evidenced by identification of the underlying molecular drivers and their regulatory molecules. During EMT, cellular reprogramming is orchestrated by core EMT transcription factors (EMT-TFs), namely ZEB1/2, TWIST1/2, SNAI1 (SNAIL) and SNAI2 (SLUG). While microRNAs have been clearly defined as regulators of EMT, the role of long non-coding RNAs (lncRNAs) in EMT is poorly defined and controversial. Determining the role of lncRNAs in EMT remains a challenge, because they are involved in a number of cellular pathways and are operating through various mechanisms. Adding to the complexity, some lncRNAs have controversial functions across different tumor types, acting as EMT promotors in some tumors and as EMT suppressors in others. The aim of this review is to summarize the role of lncRNAs involved in the regulation of EMT-TFs in human CRC. Additional candidate lncRNAs were identified through a bioinformatics analysis.

Expression of Fibrosis-Related Genes in Liver and Kidney Fibrosis in Comparison to Inflammatory Bowel Diseases.

Fibrosis is an important feature of inflammatory bowel diseases (IBD), but its pathogenesis is incompletely understood. Our aim was to identify genes important for fibrosis in IBD by comparison with kidney and liver fibrosis. First, we performed bioinformatics analysis of Gene Expression Omnibus datasets of liver and kidney fibrosis and identified CXCL9, THBS2, MGP, PTPRC, CD52, GZMA, DPT and DCN as potentially important genes with altered expression in fibrosis. We then performed qPCR analysis of the selected genes' expression on samples of fibrotic kidney, liver, Crohn's disease (CD) with and without fibrosis and ulcerative colitis (UC), in comparison to corresponding normal tissue. We found significantly altered expression in fibrosis for all selected genes. A significant difference for some genes was observed in CD with fibrosis in comparison to CD without fibrosis and UC. We conclude that similar changes in the expression of selected genes in liver, kidney fibrosis and IBD provide further evidence that fibrosis in IBD might share common mechanisms with other organs, supporting the hypothesis that fibrosis is the common pathway in diseases of various organs. Some genes were already active in IBD with inflammation without fibrosis, suggesting the early activation of profibrotic pathways or overlapping function in fibrosis and inflammation.

Metastatic EMT Phenotype Is Governed by MicroRNA-200-Mediated Competing Endogenous RNA Networks.

Epithelial-mesenchymal transition (EMT) is a fundamental physiologically relevant process that occurs during morphogenesis and organ development. In a pathological setting, the transition from epithelial toward mesenchymal cell phenotype is hijacked by cancer cells, allowing uncontrolled metastatic dissemination. The competing endogenous RNA (ceRNA) hypothesis proposes a competitive environment resembling a large-scale regulatory network of gene expression circuits where alterations in the expression of both protein-coding and non-coding genes can make relevant contributions to EMT progression in cancer. The complex regulatory diversity is exerted through an array of diverse epigenetic factors, reaching beyond the transcriptional control that was previously thought to single-handedly govern metastatic dissemination. The present review aims to unravel the competitive relationships between naturally occurring ceRNA transcripts for the shared pool of the miRNA-200 family, which play a pivotal role in EMT related to cancer dissemination. Upon acquiring more knowledge and clinical evidence on non-genetic factors affecting neoplasia, modulation of the expression levels of diverse ceRNAs may allow for the development of novel prognostic/diagnostic markers and reveal potential targets for the disruption of cancer-related EMT.

Expression of Cytokine-Coding Genes BMP8B, LEFTY1 and INSL5 Could Distinguish between Ulcerative Colitis and Crohn's Disease.

Ulcerative colitis (UC) and Crohn's disease (CD) are characterized by an imbalance between pro-inflammatory and anti-inflammatory cytokines, interfering with the resolution of inflammation. Due to the crucial role of cytokines, new insights into their profiles in UC and CD would help to improve our understanding of pathogenesis and enable the development of new treatment modalities. We provide an expression profile of cytokines in UC and CD, using bioinformatics approach, and experimental validation of expression of the selected genes. We retrieved data and analyzed the cytokine gene expression profiles of UC and CD. From ten genes with inverse expression, common to CD and UC, BMP8B, LEFTY1 and INSL5 were selected for gene expression experimental validation. Experimentally, BMP8B and INSL5 were down-regulated in both CD and UC but followed the bioinformatics trend. The expression of genes LEFTY1 and BMP8B was statistically significant when comparing UC and CD in colon and the expression of gene LEFTY1 showed statistical significance when CD in ileum and colon were compared. Using the bioinformatics approach and experimental validation, we found differences in expression profiles between UC and CD for INSL5, LEFTY1 and BMP8B. These three promising candidate genes need to be further explored at different levels, such as DNA methylation and protein expression, to provide more evidence on their potential diagnostic role in CD and UC.

Hypermethylated promoters of genes UNC5D and KCNA1 as potential novel diagnostic biomarkers in colorectal cancer.

Aim: Identification of aberrant hypermethylation in promoter regions of candidate genes to discover potential biomarkers for colorectal cancer. Materials & Methods: Genes BMP2, IRF4, KCNA1, LRRC7, NRG3, SLC27A6 and UNC5D were pre-selected in a bioinformatics study for their hypermethylation status in colorectal cancer. Methylation analysis was performed on 202 cancer tissue specimens to validate candidate genes. Results: Genes KCNA1 and UNC5D displayed methylation in 95.3 and 99.7% of The Cancer Genome Atlas dataset samples and in 96 and 98% of our experimentally tested samples, respectively. Conclusion:KCNA1 and UNC5D promoter hypermethylation holds diagnostic biomarker potential in patients with early colorectal cancer.

Differential expression of extracellular matrix­related genes DCN, EPHA4, FN1, SPARC, SPON2 and SPP1 in colorectal carcinogenesis.

During colorectal carcinogenesis, a spectrum of lesions is formed with different malignant potentials and occasionally with ambiguous morphologic features. To identify novel biomarkers, we previously performed a bioinformatics study to detect differentially expressed genes between colorectal adenoma (CRA) and colorectal carcinoma (CRC). The present study validated the diagnostic and prognostic significance of 6 components of the extracellular matrix (ECM) that were identified in the previous bioinformatics analysis [decorin (DCN), erythropoietin­producing hepatoma receptor A4 (EPHA4), fibronectin 1 (FN1), secreted protein acidic and cysteine rich (SPARC), spondin 2 (SPON2) and secreted phosphoprotein 1 (SPP1)], by analyzing their gene and protein expression levels in all samples. A total of 60 formalin­fixed paraffin­embedded biopsy samples were included in the present study, from 40 patients with CRA, malignant polyps and CRC with and without lymph node metastases. The expression of the ECM­related genes was evaluated on the mRNA level using reverse transcription­quantitative PCR (RT­qPCR) and on the protein level using immunohistochemistry. RT­qPCR results revealed differential expression among the groups for all genes, except for EPHA4. Their expression proportionally increased with the level of malignancy. Among them, SPARC, SPON2 and SPP1 were differentially expressed between CRA and malignant polyps. Immunohistochemistry analysis revealed two patterns: Positive staining of SPON2 and SPP1 in epithelial cells of healthy mucosa and dysplastic glands of CRA and CRC, and positive staining of DCN and SPARC in the lamina propria in healthy mucosa and stroma of CRA and CRC. The intensity of staining increased with the severity of lesions. The present results suggested that ECM­related proteins may have an important role in the development of CRC, with a possible diagnostic use for differentiation among various lesions, such as between CRA and malignant polyps. However, no significant potential was detected for these genes to predict lymph node metastases in CRC.

The integrative knowledge base for miRNA-mRNA expression in colorectal cancer.

"miRNA colorectal cancer" (https://mirna-coadread.omics.si/) is a freely available web application for studying microRNA and mRNA expression and their correlation in colorectal cancer. To the best of our knowledge, "miRNA colorectal cancer" has the largest knowledge base of miRNA-target gene expressions and correlations in colorectal cancer, based on the largest available sample size from the same source of data. Data from high-throughput molecular profiling of 295 colon and rectum adenocarcinoma samples from The Cancer Genome Atlas was analyzed and integrated into our knowledge base. The objective of developing this web application was to help researchers to discover the behavior and role of miRNA-target gene interactions in colorectal cancer. For this purpose, results of differential expression and correlation analyses of miRNA and mRNA data collected in our knowledge base are available through web forms. To validate our knowledge base experimentally, we selected genes FN1, TGFB2, RND3, ZEB1 and ZEB2 and miRNAs hsa-miR-200a/b/c-3p, hsa-miR-141-3p and hsa-miR-429. Both approaches revealed a negative correlation between miRNA hsa-miR-200b/c-3p and its target gene FN1 and between hsa-miR-200a-3p and its target TGFB2, thus supporting the usefulness of the developed knowledge base.