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.

Tumour budding and poorly differentiated clusters in colon cancer - different manifestations of partial epithelial-mesenchymal transition.

Morphological features including infiltrative growth, tumour budding (TB), and poorly differentiated clusters (PDCs) have a firmly established negative predictive value in colorectal cancer (CRC). Despite extensive research, the mechanisms underlying different tumour growth patterns remain poorly understood. The aim of this study was to investigate the involvement of epithelial-mesenchymal transition (EMT) in TB and PDCs in CRC. Using laser-capture microdissection, we obtained distinct parts of the primary CRC including TB, PDCs, expansive tumour front, and the central part of the tumour, and analysed the expression of EMT-related markers, i.e. the miR-200 family, ZEB1/2, RND3, and CDH1. In TB, the miR-200 family and CDH1 were significantly downregulated, while ZEB2 was significantly upregulated. In PDCs, miR-141, miR-200c, and CDH1 were significantly downregulated. No significant differences were observed in the expression of any EMT-related markers between the expansive tumour front and the central part of the tumour. Our results suggest that both TB and PDCs are related to partial EMT. Discrete differences in morphology and expression of EMT-related markers between TB and PDCs indicate that they represent different manifestations of partial EMT. TB seems to be closer to complete EMT than PDCs. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Autologous Mesenchymal Stem Cells for Treatment of Chronic Active Antibody-Mediated Kidney Graft Rejection: Report of the Phase I/II Clinical Trial Case Series.

Mesenchymal stem cell (MSCs) therapy has already been studied in kidney transplant recipients (KTRs), and the available data showed that it is safe and well tolerated. The aim of this study was to evaluate the safety and efficacy of autologous MSCs in combination with standard therapy in KTRs with biopsy-proven chronic active antibody-mediated rejection (AMR). Patients with biopsy-proven chronic active AMR received treatment with autologous bone marrow-derived MSCs (3 × 106 cells/kg iv) after completion of standard therapy and were followed for up to 12 months. The primary endpoints were safety by assessment of adverse events. Secondary endpoints included assessment of kidney graft function, immunological and histological changes related to AMR activity and chronicity assessed by conventional microscopy and molecular transcripts. A total of 3 patients were enrolled in the study before it was terminated prematurely because of adverse events. We found that AMR did not improve in any of the patients after treatment with MSCs. In addition, serious adverse events were observed in one case when autologous MSCs therapy was administered in the late phase after kidney transplantation, which requires further elucidation.

Bioinformatics Analysis of RNA-seq Data Reveals Genes Related to Cancer Stem Cells in Colorectal Cancerogenesis.

Cancer stem cells (CSC) play one of the crucial roles in the pathogenesis of various cancers, including colorectal cancer (CRC). Although great efforts have been made regarding our understanding of the cancerogenesis of CRC, CSC involvement in CRC development is still poorly understood. Using bioinformatics and RNA-seq data of normal mucosa, colorectal adenoma, and carcinoma (n = 106) from GEO and TCGA, we identified candidate CSC genes and analyzed pathway enrichment analysis (PEI) and protein-protein interaction analysis (PPI). Identified CSC-related genes were validated using qPCR and tissue samples from 47 patients with adenoma, adenoma with early carcinoma, and carcinoma without and with lymph node metastasis and were compared to normal mucosa. Six CSC-related genes were identified: ANLN, CDK1, ECT2, PDGFD, TNC, and TNXB. ANLN, CDK1, ECT2, and TNC were differentially expressed between adenoma and adenoma with early carcinoma. TNC was differentially expressed in CRC without lymph node metastases whereas ANLN, CDK1, and PDGFD were differentially expressed in CRC with lymph node metastases compared to normal mucosa. ANLN and PDGFD were differentially expressed between carcinoma without and with lymph node metastasis. Our study identified and validated CSC-related genes that might be involved in early stages of CRC development (ANLN, CDK1, ECT2, TNC) and in development of metastasis (ANLN, PDGFD).

Renal Tissue miRNA Expression Profiles in ANCA-Associated Vasculitis-A Comparative Analysis.

Anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) comprises autoimmune disease entities that cause target organ damage due to relapsing-remitting small vessel necrotizing vasculitis, and which affects various vascular beds. The pathogenesis of AAV is incompletely understood, which translates to considerable disease- and treatment-related morbidity and mortality. Recent advances have implicated microRNAs (miRNAs) in AAV; however, their accurate characterization in renal tissue is lacking. The goal of this study was to identify the intrarenal miRNA expression profile in AAV relative to healthy, non-inflammatory and inflammatory controls to identify candidate-specific miRNAs. Formalin-fixed, paraffin-embedded renal biopsy tissue samples from 85 patients were obtained. Comprehensive miRNA expression profiles were performed using panels with 752 miRNAs and revealed 17 miRNA that differentiated AAV from both controls. Identified miRNAs were annotated to characterize their involvement in pathways and to define their targets. A considerable subset of differentially expressed miRNAs was related to macrophage and lymphocyte polarization and cytokines previously deemed important in AAV pathogenesis, lending credence to the obtained results. Interestingly, several members of the miR-30 family were detected. However, a validation study of these differentially expressed miRNAs in an independent, larger sample cohort is needed to establish their potential diagnostic utility.

SINGLE NUCLEOTIDE POLYMORPHISMS IN RETINAL DETACHMENT PATIENTS WITH AND WITHOUT PROLIFERATIVE VITREORETINOPATHY.

PURPOSE: To investigate differences in genotype distributions of single nucleotide polymorphisms within genes, encoding inflammatory mediators, among patients with rhegmatogenous retinal detachment (RRD) and patients with proliferative vitreoretinopathy (PVR). METHODS: A genetic association study was performed on 191 Slovenian patients, divided into 2 groups: 113 RRD patients with PVR and 78 RRD patients without PVR. Genotype distributions were investigated within the following 13 single nucleotide polymorphisms: rs3760396 (CCL2), rs9990554 (FGF2), rs17561 (IL1A), rs2069763 (IL2), rs1800795 (IL6), rs1800871 (IL10), rs3008 (JAK3), rs2229094 (LTA), rs1042522 (TP53), rs7656613 (PDGFRA), rs7226855 (SMAD7), rs1800471 (TGFB1), and rs1800629 (TNF). RESULTS: Differences in genotype distributions between patients with RRD with or without PVR were detected in rs1800795 (IL6) (P = 0.04), rs1800871 (in the vicinity of the IL10) (P = 0.034), and rs1800471 (TGFB1) (P = 0.032). After adjustment none of the 13 analyzed single nucleotide polymorphisms showed statistically significant associations in single nucleotide polymorphism genotype distributions between patients with RRD with and without PVR. CONCLUSION: Further research is needed, particularly expanded multicentric population-based studies, to clarify the issue of genetic contribution to PVR from different genetic, clinical, and population-based aspects.

Uromodulin and microRNAs in Kidney Transplantation-Association with Kidney Graft Function.

Uromodulin and microRNAs (miRNAs) have recently been investigated as potential biomarkers for kidney graft associated pathology and outcome, with a special focus on biomarkers indicating specific disease processes and kidney graft survival. The study's aim was to determine whether expression of serum uromodulin concentration and selected miRNAs might be related to renal function in kidney transplant recipients (KTRs). The uromodulin concentration and expression of six selected miRNAs (miR-29c, miR-126, miR-146a, miR-150, miR-155, and miR-223) were determined in the serum of 100 KTRs with stable graft function and chronic kidney disease of all five stages. Kidney graft function was estimated with routine parameters (creatinine, urea, cystatin C, and Chronic Kidney Disease Epidemiology Collaboration study equations) and precisely measured using chromium-51 labelled ethylenediaminetetraacetic-acid clearance. The selected miRNAs were shown to be independent of kidney graft function, indicating their potential as biomarkers of associated kidney graft disease processes. In contrast, the serum uromodulin level depended entirely on kidney graft function and thus reflected functioning tubules rather than any specific kidney graft injury. However, decreased concentrations of serum uromodulin can be observed in the early course of tubulointerstitial injury, thereby suggesting its useful role as an accurate, noninvasive biomarker of early (subclinical) kidney graft injury.

Sclerosing Melanocytic Lesions (sclerosing Melanomas with Nevoid Features and Sclerosing Nevi with Pseudomelanomatous Features) - An Analysis of 90 Lesions.

BACKGROUND: Sclerosing melanocytic lesions, which are characterized by either focal or diffuse sclerosis in the dermal component and atypical proliferation of predominantly nevoid melanocytes, remain poorly defined. Our aim was to analyze systematically their morphologic spectrum, especially the distinction between sclerosing melanocytic nevus and sclerosing melanoma, which has not been well documented. PATIENTS AND METHODS: We collected 90 sclerosing melanocytic lesions, occurring in 82 patients (49 male, 33 female; age range from 21 to 89 years). A four probe fluorescent in situ hybridization (FISH) assay was performed in 41 lesions to substantiate the diagnosis of sclerosing melanomas. RESULTS: A prominent full-thickness pagetoid spread of melanocytes was identified in 44 (48%) lesions, and a melanoma in situ adjacent to the sclerosis in 55 (61%) lesions. In the intrasclerotic component, maturation was absent in 40 (44%) and mitotic figures were identified in 18 (20%) lesions. Of the 90 lesions, 26 (29%) were diagnosed morphologically as nevi and 64 (71%) as melanomas (Breslow thickness from 0.4 to 1.8 mm), including 45 (50%) melanomas with an adjacent nevus. A four-probe FISH assay was positive in the sclerotic component in 14 of 25 lesions diagnosed morphologically as melanomas and none of 16 nevi. A sentinel lymph node biopsy was performed for 17 lesions and was negative in all cases. CONCLUSIONS: Sclerosing melanocytic lesions form a morphologic spectrum and include both nevi and melanomas. The pathogenesis of sclerosis remains obscure but seems to be induced by melanocytes or an unusual host response in at least a subset of lesions.

Down-regulation of microRNAs of the miR-200 family and up-regulation of Snail and Slug in inflammatory bowel diseases - hallmark of epithelial-mesenchymal transition.

Fibrosis is an important feature of inflammatory bowel diseases (IBD), particularly Crohn's disease (CD), but its pathogenesis is poorly understood. To determine the postulated involvement of epithelial-mesenchymal transition (EMT) in the development of fibrosis in IBD, we analysed the expression profiles of the miR-200 family which has been shown to induce EMT in experimental models and various human diseases. We also analysed the expression of Snail and Slug, postulated targets of the investigated microRNAs. Ten patients with ulcerative colitis (UC) and 10 patients with CD who underwent colon resection were included. From each, two tissue samples were chosen (one with the most severely and one with the least affected or normal mucosa) for analysis of microRNAs expression using real-time polymerase chain reaction, and Snail and Slug expression using immunohistochemistry. We found significant down-regulation of all investigated microRNAs in CD, and of three investigated microRNAs in UC, in comparison to the normal or the least affected mucosa. Comparing UC and CD, four microRNAs were significantly more down-regulated in CD than in UC. Snail and Slug were expressed in the injured epithelium and occasionally in mesothelial cells and submesothelial fibroblasts. Our finding of down-regulation of the miR-200 family and up-regulation of transcription repressors Snail and Slug supports the postulated role of EMT in the pathogenesis of fibrosis in IBD. The described expression patterns are consistent with the notion that fibrosis does not occur only in CD but also in UC, being much more severe in CD.

Long-term survival in glioblastoma: methyl guanine methyl transferase (MGMT) promoter methylation as independent favourable prognostic factor.

BACKGROUND: In spite of significant improvement after multi-modality treatment, prognosis of most patients with glioblastoma remains poor. Standard clinical prognostic factors (age, gender, extent of surgery and performance status) do not clearly predict long-term survival. The aim of this case-control study was to evaluate immuno-histochemical and genetic characteristics of the tumour as additional prognostic factors in glioblastoma. PATIENTS AND METHODS: Long-term survivor group were 40 patients with glioblastoma with survival longer than 30 months. Control group were 40 patients with shorter survival and matched to the long-term survivor group according to the clinical prognostic factors. All patients underwent multimodality treatment with surgery, postoperative conformal radiotherapy and temozolomide during and after radiotherapy. Biopsy samples were tested for the methylation of MGMT promoter (with methylation specific polymerase chain reaction), IDH1 (with immunohistochemistry), IDH2, CDKN2A and CDKN2B (with multiplex ligation-dependent probe amplification), and 1p and 19q mutations (with fluorescent in situ hybridization). RESULTS: Methylation of MGMT promoter was found in 95% and in 36% in the long-term survivor and control groups, respectively (p < 0.001). IDH1 R132H mutated patients had a non-significant lower risk of dying from glioblastoma (p = 0.437), in comparison to patients without this mutation. Other mutations were rare, with no significant difference between the two groups. CONCLUSIONS: Molecular and genetic testing offers additional prognostic and predictive information for patients with glioblastoma. The most important finding of our analysis is that in the absence of MGMT promoter methylation, longterm survival is very rare. For patients without this mutation, alternative treatments should be explored.

Selection and Evaluation of mRNA and miRNA Reference Genes for Expression Studies (qPCR) in Archived Formalin-Fixed and Paraffin-Embedded (FFPE) Colon Samples of DSS-Induced Colitis Mouse Model.

The choice of appropriate reference genes is essential for correctly interpreting qPCR data and results. However, the majority of animal studies use a single reference gene without any prior evaluation. Therefore, many qPCR results from rodent studies can be misleading, affecting not only reproducibility but also translatability. In this study, the expression stability of reference genes for mRNA and miRNA in archived FFPE samples of 117 C57BL/6JOlaHsd mice (males and females) from 9 colitis experiments (dextran sulfate sodium; DSS) were evaluated and their expression analysis was performed. In addition, we investigated whether normalization reduced/neutralized the influence of inter/intra-experimental factors which we systematically included in the study. Two statistical algorithms (NormFinder and Bestkeeper) were used to determine the stability of reference genes. Multivariate analysis was made to evaluate the influence of normalization with different reference genes on target gene expression in regard to inter/intra-experimental factors. Results show that archived FFPE samples are a reliable source of RNA and imply that the FFPE procedure does not change the ranking of stability of reference genes obtained in fresh tissues. Multivariate analysis showed that the histological picture is an important factor affecting the expression levels of target genes.

Analyzing the invasive front of colorectal cancer - By punching tissue block or laser capture microdissection?

The aim of this study was to determine the advantages and limitations of two commonly used sampling techniques, i.e., punching tissue block (PTB) and laser capture microdissection (LCM) when investigating tumor cell-derived gene expression patterns at the invasive front of colorectal cancer (CRC). We obtained samples from 20 surgically removed CRCs at locations crucial for tumor progression, i.e., the central part, the expansive front and the infiltrative front exhibiting tumor budding (TB), using both sampling techniques. At each location, we separately analyzed the expressions of miR-200 family (miR-141, miR-200a, miR-200b, miR-200c and miR-429), known as reliable markers of epithelial-mesenchymal transition (EMT). We found significant downregulation of all members of miR-200 family at the infiltrative front in comparison to the central part regardless of the used sampling technique. However, when comparing miR-200 expression between the expansive and the infiltrative front, we found significant downregulation of all tested miR-200 at the infiltrative front only in samples obtained by LCM. Our results suggest that, PTB is an adequate technique for studying the differences in tumor gene expression between the central part and the invasive front of CRC, but is insufficient to analyze and compare morphologically distinct patterns along the invasive front including TB. For this purpose, the use of LCM is essential.

Tissue miRNA profile is associated with acute tubular necrosis, rejection phenotypes and BK polyomavirus-associated nephropathy in human kidney allografts.

INTRODUCTION: MicroRNAs, short noncoding RNAs, are involved in the modulation of gene expression, mainly by inhibiting the translation of mRNAs. Under physiological conditions, miRNAs are involved in viral infections and immune responses, among others; aberrant miRNA expression has been associated with kidney transplant pathologies, but a comprehensive comparison of later, particularly in tissue sections, is still pending. METHODS: We used the genome-wide screening of miRNAs to identify those potentially involved in the disease processes after kidney transplantation. RNA was isolated from formalin-fixed paraffin- embedded kidney biopsy samples. Study included 8 patients with acute tubular necrosis (ATN), 8 patients with antibody-mediated rejection (ABMR), 10 patients with T-cell-mediated rejection (TCMR), 10 patients with BK polyomavirus-associated nephropathy (BKPyVAN) and 12 surveillance biopsies from patients with stable allograft function and no major abnormalities (normal allografts, CTRL). RESULTS: We found 136 miRNAs differentially expressed in diseased kidney transplant tissue compared with normal allografts; of these, 74 miRNAs were differentially expressed in ABMR, 65 in ATN, 62 in BKPyVAN, 69 in TCMR, and 16 miRNAs were not associated with a specific disease phenotype. In addition, 29 miRNAs were differently expressed between ABMR and ATN, 39 between BKPyVAN and TCMR, and 20 between BKPyVAN and ABMR, and 38 between ABMR and TCMR. CONCLUSION: Our findings show that miRNA derived from kidney allograft biopsy samples represent an additional diagnostic tool to distinguish different disease phenotypes. This finding has the potential to assist clinicians in therapeutic decision making and to translate to non-invasive monitoring of patients, e.g. blood samples.

MicroRNAs and cardiac sarcoplasmic reticulum calcium ATPase-2 in human myocardial infarction: expression and bioinformatic analysis.

BACKGROUND: Cardiac sarco(endo)plasmic reticulum calcium ATPase-2 (SERCA2) plays one of the central roles in myocardial contractility. Both, SERCA2 mRNA and protein are reduced in myocardial infarction (MI), but the correlation has not been always observed. MicroRNAs (miRNAs) act by targeting 3'-UTR mRNA, causing translational repression in physiological and pathological conditions, including cardiovascular diseases. One of the aims of our study was to identify miRNAs that could influence SERCA2 expression in human MI. RESULTS: The protein SERCA2 was decreased and 43 miRNAs were deregulated in infarcted myocardium compared to corresponding remote myocardium, analyzed by western blot and microRNA microarrays, respectively. All the samples were stored as FFPE tissue and in RNAlater. miRNAs binding prediction to SERCA2 including four prediction algorithms (TargetScan, PicTar, miRanda and mirTarget2) identified 213 putative miRNAs. TAM and miRNApath annotation of deregulated miRNAs identified 18 functional and 21 diseased states related to heart diseases, and association of the half of the deregulated miRNAs to SERCA2. Free-energy of binding and flanking regions (RNA22, RNAfold) was calculated for 10 up-regulated miRNAs from microarray analysis (miR-122, miR-320a/b/c/d, miR-574-3p/-5p, miR-199a, miR-140, and miR-483), and nine miRNAs deregulated from microarray analysis were used for validation with qPCR (miR-21, miR-122, miR-126, miR-1, miR-133, miR-125a/b, and miR-98). Based on qPCR results, the comparison between FFPE and RNAlater stored tissue samples, between Sybr Green and TaqMan approaches, as well as between different reference genes were also performed. CONCLUSION: Combing all the results, we identified certain miRNAs as potential regulators of SERCA2; however, further functional studies are needed for verification. Using qPCR, we confirmed deregulation of nine miRNAs in human MI, and show that qPCR normalization strategy is important for the outcome of miRNA expression analysis in human MI.

Differential expression of microRNAs miR-21, miR-31, miR-203, miR-125a-5p and miR-125b and proteins PTEN and p63 in verrucous carcinoma of the head and neck.

AIMS: To investigate the expression of microRNAs miR-21, miR-31, miR-203, miR-125a-5p and miR-125b and proteins phosphatase and tensin homologue (PTEN) and p63 in verrucous carcinoma (VC) of the head and neck. METHODS AND RESULTS: Thirty cases of VC, 50 cases of conventional squamous cell carcinoma (SCC) and 30 samples of normal epithelium of the head and neck were included. Real-time polymerase chain reaction and immunohistochemistry were used to analyse the expression of microRNAs and proteins, respectively. In comparison to normal epithelium, miR-21 was overexpressed in both VC and SCC and miR-31 was overexpressed in VC and in well- and moderately differentiated SCC. Levels of miR-203 were elevated in VC but unaltered or reduced in SCC, and levels of miR-125a-5p and miR-125b were reduced in VC but unaltered in SCC. PTEN was down-regulated in both VC and SCC, whereas p63 was down-regulated in VC but up-regulated in SCC. Differential expression of p63 in VC correlated inversely with the expression of miR-21 and miR-203. CONCLUSIONS: Differences between VC, SCC and normal epithelium in expression profiles of investigated molecules indicate their association with the pathogenesis and clinicopathological characteristics of VC. Our results suggest that some microRNAs and proteins, particularly miR-125b, miR-203 and p63, might be useful in the diagnosis of VC.

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.

Intra­tumor heterogeneity of cancer stem cell­related genes and their potential regulatory microRNAs in metastasizing colorectal carcinoma.

Intra­tumor heterogeneity (ITH) is related to cancer progression, therapy resistance and recurrences, and is one of the challenging fields in cancerogenesis research. Cancer stem cells (CSC) are thought to be crucially involved in the pathogenesis of several cancer types, including colorectal carcinoma (CRC), and associated with ITH. In the present study, the expression gradient of four genes related to CSC (L1TD1, SLITRK6, ST6GALNAC1 and TCEA3) and their potential regulatory microRNAs (miRNAs) were investigated in the central part and invasive front of the primary tumor, as well as in lymph node and liver metastases. In total, 63 formalin­fixed paraffin­embedded biopsy samples of primary tumor (central part, invasive tumor front), as well as lymph node and liver metastases from 19 patients with CRC, were analyzed. The expression of selected genes (L1TD1, SLITRK6, ST6GALNAC1 and TCEA3) and miRNAs (miR­199a­3p, miR­425­5p, miR­1225­3p, miR­1233­3p and miR­1303) was evaluated using reverse transcription­quantitative PCR. Significant differences in expression were identified for all investigated genes in lymph node metastasis, but not in the liver metastases. All investigated miRNAs were significantly differentially expressed in lymph node metastasis, and miR­199a­3p, miR­425­5p and miR­1233­3p in liver metastasis. Furthermore, a negative correlation between the expression of miR­199a­3p and expression of its potential target gene SLITRK6 was confirmed. The present results provide further evidence that expression of CSC­related genes and their potential regulatory miRNAs contribute to ITH in CRC, lymph node and liver metastasis. The SLITRK6 gene and its regulatory miRNA miR­199a­3p are promising for further validation in functional studies to deepen the present understanding of the regulation of CSC­related genes in CRC.

Differential Expression of Decorin in Metastasising Colorectal Carcinoma Is Regulated by miR-200c and Long Non-Coding RNAs.

Decorin (DCN) is one of the matricellular proteins that participate in normal cells' function as well as in cancerogenesis. While its expression in primary tumours is well known, there is limited data about its expression in metastases. Furthermore, the post-transcriptional regulation of DCN is still questionable, although it is well accepted that it is an important mechanism of developing metastatic cancer. The aim of our study was to analyse the expression of DCN and its potential regulatory ncRNAs in metastatic colorectal carcinoma (CRC). Nineteen patients with metastatic CRC were included. Using qPCR, we analysed the expression of DCN, miR-200c and five lncRNAs (LUCAT1, MALAT1, lncTCF7, XIST, and ZFAS1) in lymph node and liver metastases in comparison to the invasive front and central part of a primary tumour. Our results showed insignificant upregulation of DCN and significant upregulation for miR-200c, MALAT1, lncTCF7 and ZFAS1 in metastases compared to the primary tumour. miR-200c showed a positive correlation with DCN, and the aforementioned lncRNAs exhibited a significant positive correlation with miR-200c expression in metastatic CRC. Our results suggest that DCN as well as miR-200c, MALAT1, lncTCF7 and ZFAS1 contribute to the development of metastases in CRC and that regulation of DCN expression in CRC by ncRNAs is accomplished in an indirect manner.

miR-200b, ZEB2 and PTPN13 Are Downregulated in Colorectal Carcinoma with Serosal Invasion.

Serosal invasion is an independent negative prognostic factor in certain cancers, including CRC. However, the mechanisms behind serosal invasion are poorly understood. We therefore assumed that epithelial-mesenchymal transition (EMT) might be involved. Our study included 34 patients with CRC, 3 stage pT2, 14 stage pT3 and 17 showing serosal invasion (stage pT4a according to TNM staging system). RNA isolated from formalin-fixed paraffin-embedded tissue samples was analysed for expression of the miR-200 family and their target genes CDKN1B, ONECUT2, PTPN13, RND3, SOX2, TGFB2 and ZEB2 using real-time PCR. We found upregulation of miR-200b and ONECUT2 in CRC pT3 and pT4a compared to normal mucosa, and downregulation of CDKN1B in CRC pT3. Moreover, we observed, downregulation of miR-200b, PTPN13 and ZEB2 in CRC with serosal invasion (pT4a) compared to pT3. Our results suggest the involvement of partial EMT in serosal invasion of CRC. In addition, PTPN13 seems to be one of the important regulators involved in serosal invasion, and ONECUT2 in tumour growth.

The Role of Immune-Related miRNAs in the Pathology of Kidney Transplantation.

MicroRNAs (miRNAs) are members of the non-coding regulatory RNA family that play pivotal roles in physiological and pathological conditions, including immune response. They are particularly interesting as promising therapeutic targets, prognostic and diagnostic markers due to their easy detection in body fluids and stability. There is accumulating evidence that different miRNAs provide disease-specific signatures in liquid samples of distinct kidney injuries. Using experimental models and human samples, there have been numerous suggestions that immune-related miRNAs are also important contributors to the development of different kidney diseases as well as important markers for monitoring response after kidney transplantation. However, there are limited data for understanding their function in the molecular pathways of allograft pathologies. In our review, we focused on microRNAs that are related to different aspects of immune response after kidney transplantation.