A novel mutation of congenital nephrotic syndrome in a Slovenian child eventually receiving a renal transplant

Congenital nephrotic syndrome (CNS) is a rare disease defined as heavy proteinuria, hypoalbuminemia, hyperlipidemia, and edema presenting in the first three months of life. It is most commonly caused by mutations in the NPHS1 gene associated with nephrotic syndrome type 1, also known as Finnish-type CNS, which is inherited in an autosomal recessive manner. Symptomatic treatment with intravenous albumins, vitamins, minerals, nutritional, and hormonal supplementation and treatment of complications are mandatory. Children refractory to the symptomatic treatment are recommended to undergo nephrectomy and renal replacement therapy, preferably renal transplantation. We report on a child with Finnish type CNS with a NPHS1 mutation, which is the first case confirmed by genetic study in Slovenia. We showed for the first time that homozygous mutation c.2928-3del in the NPHS1 gene caused exon 22 skipping, leading to a truncated protein and Fin-minor phenotype

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

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)

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

Identification and validation of new cancer stem cell-related genes and their regulatory microRNAs in colorectal cancerogenesis

Significant progress has been made in the last decade in our understanding of the pathogenetic mechanisms of colorectal cancer (CRC). Cancer stem cells (CSC) have gained much attention and are now believed to play a crucial role in the pathogenesis of various cancers, including CRC. In the current study, we validated gene expression of four genes related to CSC, L1TD1, SLITRK6, ST6GALNAC1 and TCEA3, identified in a previous bioinformatics analysis. Using bioinformatics, potential miRNA-target gene correlations were prioritized. In total, 70 formalin-fixed paraffin-embedded biopsy samples from 47 patients with adenoma, adenoma with early carcinoma and CRC without and with lymph node metastases were included. The expression of selected genes and microRNAs (miRNAs) was evaluated using quantitative PCR. Differential expression of all investigated genes and four of six prioritized miRNAs (hsa-miR-199a-3p, hsa-miR-335-5p, hsa-miR-425-5p, hsa-miR-1225-3p, hsa-miR-1233-3p and hsa-miR-1303) was found in at least one group of CRC cancerogenesis. L1TD1, SLITRK6, miR-1233-3p and miR-1225-3p were correlated to the level of malignancy. A negative correlation between miR-199a-3p and its predicted target SLITRK6 was observed, showing potential for further experimental validation in CRC. Our results provide further evidence that CSC-related genes and their regulatory miRNAs are involved in CRC development and progression and suggest that some them, particularly miR-199a-3p and its SLITRK6 target gene, are promising for further validation in CRC

Expression of NANOG and Its Regulation in Oral Squamous Cell Carcinoma

Background. Results of previous studies suggest that NANOG may be an important prognostic biomarker in oral squamous cell carcinoma (OSCC), but there are contradictory results regarding NANOG expression patterns on mRNA and protein levels, and the mechanisms of its regulation are poorly understood. Our aim was to analyze the expression and diagnostic significance of NANOG in OSCC, and the possible mechanisms of its regulation, i.e., protein regulators on mRNA level (OCT4, SOX2, KLF4, AGR2, and NOTCH1), methylation status, copy number variation, and regulatory miRNAs, miR-145, miR-335, miR-150, miR-34a, miR-128, and miR-27a. Methods. Our study included 120 patients with OSCC. Expression of NANOG protein and mRNA was analyzed using immunohistochemistry and qPCR. Expression of regulatory factors, miRNAs, and copy number variation was performed using qPCR. Methylation status of NANOG promoter was determined using PCR and Sanger sequencing. Results. We detected upregulation of NANOG and OCT4 and downregulation of NOTCH1 and AGR2 mRNA in OSCC with lymph node metastases compared to OSCC without lymph node metastases. We observed a strong positive correlation between mRNAs of NANOG and those of its protein regulators OCT4, SOX2, NOTCH1, AGR2, and KLF4. The expression of NANOG was in positive correlation with the expression of miR-34a. There was also a correlation between T status of OSCC and the expression of miR-335 and miR-150 and a correlation of miR-150 with the N status of T2 OSCC. NANOG promoter methylation and copy number variation were only observed in a small proportion of samples. Conclusions. Our findings confirm the diagnostic significance of NANOG in OSCC and provide information on NANOG expression patterns on both mRNA and protein levels. They also suggest that protein regulators and microRNAs might play a crucial role, whereas methylation of its promoter and copy number variation probably have a minor role in the regulation of NANOG expression in OSCC

MicroRNAs, Innate Immunity and Ventricular Rupture in Human Myocardial Infarction

MicroRNAs are non-coding RNAs, functionioning as post-transcriptional regulators of gene expression. Some microRNAs have been demonstrated to play a role in regulation of innate immunity. After myocardial infarction (MI), innate immunity is activated leading to an acute inflammatory reaction. There is evidence that an intense inflammatory reaction might contribute to the development of ventricular rupture (VR) after MI

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

Expression of extracellular matrix-related genes and their regulatory microRNAs in problematic colorectal polyps

Colorectal carcinoma usually evolves gradually, forming a spectrum of lesions, due to accumulation of genetic mutations and epigenetic alterations. Many early lesions are detected since the introduction of screening programs. The greatest challenge is to distinguish between adenomas with epithelial misplacement (AEM) and adenomas with early carcinoma (AEC), considering the diagnosis affects prognosis and treatment. We analyzed the expression of selected extracellular matrix (ECM)-related genes and proteins, and their regulatory microRNAs using RT-qPCR and immunohistochemistry in biopsies from 44 patients. Differences were observed in AEM in comparison to AEC for DCN, EPHA4, FN1, SPON2, and SPP1, reflecting inflammatory stromal reaction to traumatisation and misplacement of dysplastic glands in the submucosa in the former, and desmoplastic stromal reaction to true invasion of dysplastic glands in the submucosa in the latter. Expression of regulatory microRNAs hsa-miR-200c and hsa-miR-146a significantly negatively correlated with the expression of their regulated genes, while significant difference between AEM and AEC was observed only for hsa-miR-29c. The described expression patterns are too complex to be used in diagnostic work, but might contribute to better understanding ECM changes in colorectal carcinoma development, helping to find new markers in the future