Expression of RASSF1A, DIRAS3, and AKAP9 Genes in Thyroid Lesions: Implications for Differential Diagnosis and Prognosis of Thyroid Carcinomas.

Thyroid carcinoma is the primary endocrine malignancy worldwide. The preoperative examination of thyroid tissue lesion is often unclear. Approximately 25% of thyroid cancers cannot be diagnosed definitively without post-surgery histopathological examination. The assessment of diagnostic and differential markers of thyroid cancers is needed to improve preoperative diagnosis and reduce unnecessary treatments. Here, we assessed the expression of RASSF1A, DIRAS3, and AKAP9 genes, and the presence of BRAF V600E point mutation in benign and malignant thyroid lesions in a Polish cohort (120 patients). We have also performed a comparative analysis of gene expression using data obtained from the Gene Expression Omnibus (GEO) database (307 samples). The expression of RASSF1A and DIRAS3 was decreased, whereas AKAP9's was increased in pathologically changed thyroid compared with normal thyroid tissue, and significantly correlated with e.g., histopathological type of lesion papillary thyroid cancer (PTC) vs follicular thyroid cancer (FTC), patient's age, tumour stage, or its encapsulation. The receiver operating characteristic (ROC) analysis for the more aggressive FTC subtype differential marker suggests value in estimating RASSF1A and AKAP9 expression, with their area under curve (AUC), specificity, and sensitivity at 0.743 (95% CI: 0.548-0.938), 82.2%, and 66.7%; for RASSF1A, and 0.848 (95% CI: 0.698-0.998), 54.8%, and 100%, for AKAP9. Our research gives new insight into the basis of the aggressiveness and progression of thyroid cancers, and provides information on potential differential markers that may improve preoperative diagnosis.

The Expression of TGF-ß1, SMAD3, ILK and miRNA-21 in the Ectopic and Eutopic Endometrium of Women with Endometriosis.

The molecular pathogenesis of endometriosis has been associated with pathological alterations of protein expression via disturbances in homeostatic genes, miRNA expression profiles, and signaling pathways that play an essential role in the epithelial-mesenchymal transition (EMT) process. TGF-ß1 has been hypothesized to play a key role in the development and progression of endometriosis, but the activation of a specific mechanism via the TGF-ß-SMAD-ILK axis in the formation of endometriotic lesions is poorly understood. The aim of this study was to assess the expression of EMT markers (TGF-ß1, SMAD3, ILK) and miR-21 in ectopic endometrium (ECE), in its eutopic (EUE) counterpart, and in the endometrium of healthy women. The expression level of the tested genes and miRNA was also evaluated in peripheral blood mononuclear cells (PBMC) in women with and without endometriosis. Fifty-four patients (n = 54; with endometriosis, n = 29, and without endometriosis, n = 25) were enrolled in the study. The expression levels (RQ) of the studied genes and miRNA were evaluated using qPCR. Endometriosis patients manifested higher TGF-ß1, SMAD3, and ILK expression levels in the eutopic endometrium and a decreased expression level in the ectopic lesions in relation to control tissue. Compared to the endometrium of healthy participants, miR-21 expression levels did not change in the eutopic endometrium of women with endometriosis, but the RQ was higher in their endometrial implants. In PBMC, negative correlations were found between the expression level of miR-21 and the studied genes, with the strongest statistically significant correlation observed between miR-21 and TGF-ß1. Our results suggest the loss of the endometrial epithelial phenotype defined by the differential expression of the TGF-ß1, SMAD3 and ILK genes in the eutopic and ectopic endometrium. We concluded that the TGF-ß1-SMAD3-ILK signaling pathway, probably via a mechanism related to the EMT, may be important in the pathogenesis of endometriosis. We also identified miR-21 as a possible inhibitor of this TGF-ß1-SMAD3-ILK axis.

Assessment of BMP7, SMAD4, and CDH1 Expression Profile and Regulatory miRNA-542-3p in Eutopic and Ectopic Endometrium of Women with Endometriosis.

Alterations in the expression of numerous genes and the miRNAs that are recognized as their regulators in the endometrial cells of women with endometriosis may disrupt the intracellular signaling pathways associated with epithelial-mesenchymal transition (EMT). So far, the functional role of BMP7 in endometrial physiology has been confirmed, especially in the context of fertility, but the role of the activation of a specific mechanism operating through the BMP-SMAD-CDH1 axis in the formation of endometrial lesions remains unexplored. The aim of this study was to evaluate the expression profile of miR-542-3p and the EMT markers (BMP7, SMAD4, CDH1) in matched eutopic endometrium (EUE) and ectopic endometrium (ECE) samples from women with endometriosis in relation to healthy women. The levels of expression of the studied genes and miRNA in peripheral blood mononuclear cells (PBMCs) obtained from women diagnosed with endometriosis and those without the disease were also evaluated. Fifty-four patients (n = 54: with endometriosis-n = 29 and without endometriosis-n = 25) were included in the study. A comparative analysis of the relative mean expression values (RQ) of the studied mRNA and miRNA assessed by RT-qPCR demonstrated downregulation of BMP7, SMAD4, and CDH1 expression in ectopic lesions and upregulation in the eutopic endometrium compared with the control group. In the eutopic tissue of women with endometriosis, miR-542-3p expression was similar to that of the control but significantly lower than in endometrial lesions. We also confirmed a trend towards a negative correlation between miR-542-3p and BMP7 in ectopic tissue, and in PBMC, a significant negative correlation of miR-542-3p with further BMP signaling genes, i.e., SMAD4 and CDH1, was observed. These results indicate that the miRNA selected by us may be a potential negative regulator of BMP7-SMAD4-CDH1 signaling associated with EMT. The different patterns of BMP7, SMAD4, and CDH1 gene expression in ECE, EUE, and the control endometrium observed by us suggests the loss of the endometrial epithelium phenotype in women with endometriosis and demonstrates their involvement in the pathogenesis and pathomechanism of this disease.

The Comparison of Serum Exosome Protein Profile in Diagnosis of NSCLC Patients.

A thorough study of the exosomal proteomic cargo may enable the identification of proteins that play an important role in cancer development. The aim of this study was to compare the protein profiles of the serum exosomes derived from non-small lung cancer (NSCLC) patients and healthy volunteers (control) using the high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS) method to identify potentially new diagnostic and/or prognostic protein biomarkers. Proteins exclusively identified in NSCLC and control groups were analyzed using several bioinformatic tools and platforms (FunRich, Vesiclepedia, STRING, and TIMER2.0) to find key protein hubs involved in NSCLC progression and the acquisition of metastatic potential. This analysis revealed 150 NSCLC proteins, which are significantly involved in osmoregulation, cell-cell adhesion, cell motility, and differentiation. Among them, 3 proteins: Interleukin-34 (IL-34), HLA class II histocompatibility antigen, DM alpha chain (HLA-DMA), and HLA class II histocompatibility antigen, DO beta chain (HLA-DOB) were shown to be significantly involved in the cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) infiltration processes. Additionally, detected proteins were analyzed according to the presence of lymph node metastasis, showing that differences in frequency of detection of protein FAM166B, killer cell immunoglobulin-like receptor 2DL1, and olfactory receptor 52R1 correlate with the N feature according to the TNM Classification of Malignant Tumors. These results prove their involvement in NSCLC lymph node spread and metastasis. However, this study requires further investigation.

Isothiocyanates (ITCs) 1-(Isothiocyanatomethyl)-4-phenylbenzene and 1-Isothiocyanato-3,5-bis(trifluoromethyl)benzene-Aldehyde Dehydrogenase (ALDH) Inhibitors, Decreases Cisplatin Tolerance and Migratory Ability of NSCLC.

One of the main treatment modalities for non-small-cell lung cancer (NSCLC) is cisplatin-based chemotherapy. However, the acquisition of cisplatin resistance remains a major problem. Existing chemotherapy regimens are often ineffective against cancer cells expressing aldehyde dehydrogenase (ALDH). As such, there is an urgent need for therapies targeting ALDH-positive cancer cells. The present study compares the anticancer properties of 36 structurally diverse isothiocyanates (ITCs) against NSCLC cells with the ALDH inhibitor disulfiram (DSF). Their potential affinity to ALDH isoforms and ABC proteins was assessed using AutoDockTools, allowing for selection of three compounds presenting the strongest affinity to all tested proteins. The selected ITCs had no impact on NSCLC cell viability (at tested concentrations), but significantly decreased the cisplatin tolerance of cisplatin-resistant variant of A549 (A549CisR) and advanced (stage 4) NSCLC cell line H1581. Furthermore, long-term supplementation with ITC 1-(isothiocyanatomethyl)-4-phenylbenzene reverses the EMT phenotype and migratory potential of A549CisR to the level presented by parental A549 cells, increasing E-Cadherin expression, followed by decreased expression of ABCC1 and ALDH3A1. Our data indicates that the ALDH inhibitors DSF and ITCs are potential adjuvants of cisplatin chemotherapy.

Effect of physical training on parathyroid hormone and bone turnover marker profile in relation to vitamin D supplementation in soccer players.

The aim of the study was to assess the impact of vitamin D supplementation and regular physical activity on 25-hydroxyvitamin D [25(OH)D], parathyroid hormone (PTH) and bone turnover marker concentrations in healthy male athletes. Twenty-five youth soccer players were divided into groups: non-supplemented (GN) and supplemented (GS) with a vitamin D dose of 20 000 IU twice a week for 8 weeks. The study was conducted during an 8-week preseason period, from mid-January to mid-March. At baseline (T1) and at the end of this period (T2), the serum concentrations of 25(OH)D, (PTH), osteocalcin (OC) and ß-isomerized C-terminal telopeptide of type I collagen (ß-CTx) were measured. At T2, 25(OH)D increased by 70% in GS (p = 0.004) and by 6% in GN (p > 0.05). Significant differences between GS and GN groups were observed throughout the study in the group-by-time interaction and changes of 25(OH)D (p = 0.002; η 2 p = 0.36) and OC (p = 0.008; η 2 p = 0.26). Increased OC (ES = 0.74; moderate) and ß-CTx (ES = 1.31, large) in GN athletes who had an optimal baseline vitamin D level (GO) were observed. In GN, at T2, ß-CTx positively correlated with PTH and OC (p = 0.007 and p = 0.002). In GS, ß-CTx positively correlated with OC at both time points (T1, p = 0.027 and T2, p = 0.037). A negative correlation between 25(OH)D and PTH was observed at T2 (p = 0.018). The obtained results suggest that the 20 000 IU vitamin D3 dose applied twice a week for 8 weeks is effective for vitamin D compensation and sufficient to maintain the correct PTH concentration, as revealed by changes in the bone marker concentrations. In conclusion, the results suggest that the applied vitamin D supplementation dose in athletes leads to intensive bone remodelling and has protective effects on bone under intensive physical effort.

Circulating miRNAs Related to Epithelial-Mesenchymal Transitions (EMT) as the New Molecular Markers in Endometriosis.

Endometriosis is a chronic gynecological disease defined by the presence of endometrial-like tissue found outside the uterus, most commonly in the peritoneal cavity. Endometriosis lesions are heterogenous but usually contain endometrial stromal cells and epithelial glands, immune cell infiltrates and are vascularized and innervated by nerves. The complex etiopathogenesis and heterogenity of the clinical symptoms, as well as the lack of a specific non-invasive diagnostic biomarkers, underline the need for more advanced diagnostic tools. Unfortunately, the contribution of environmental, hormonal and immunological factors in the disease etiology is insufficient, and the contribution of genetic/epigenetic factors is still fragmentary. Therefore, there is a need for more focused study on the molecular mechanisms of endometriosis and non-invasive diagnostic monitoring systems. MicroRNAs (miRNAs) demonstrate high stability and tissue specificity and play a significant role in modulating a range of molecular pathways, and hence may be suitable diagnostic biomarkers for the origin and development of endometriosis. Of these, the most frequently studied are those related to endometriosis, including those involved in epithelial-mesenchymal transition (EMT), whose expression is altered in plasma or endometriotic lesion biopsies; however, the results are ambiguous. Specific miRNAs expressed in endometriosis may serve as diagnostics markers with prognostic value, and they have been proposed as molecular targets for treatment. The aim of this review is to present selected miRNAs associated with EMT known to have experimentally confirmed significance, and discuss their utility as biomarkers in endometriosis.

Molecular Mechanisms of Chemoresistance Induced by Cisplatin in NSCLC Cancer Therapy.

Cancer cells utilise several mechanisms to increase their survival and progression as well as their resistance to anticancer therapy: deregulation of growth regulatory pathways by acquiring grow factor independence, immune system suppression, reducing the expression of antigens activating T lymphocyte cells (mimicry), induction of anti-apoptotic signals to counter the action of drugs, activation of several DNA repair mechanisms and driving the active efflux of drugs from the cell cytoplasm, and epigenetic regulation by microRNAs (miRNAs). Because it is commonly diagnosed late, lung cancer remains a major malignancy with a low five-year survival rate; when diagnosed, the cancer is often highly advanced, and the cancer cells may have acquired drug resistance. This review summarises the main mechanisms involved in cisplatin resistance and interactions between cisplatin-resistant cancer cells and the tumour microenvironment. It also analyses changes in the gene expression profile of cisplatin sensitive vs. cisplatin-resistant non-small cell lung cancer (NSCLC) cellular model using the GSE108214 Gene Expression Omnibus database. It describes a protein-protein interaction network that indicates highly dysregulated TP53, MDM2, and CDKN1A genes as they encode the top networking proteins that may be involved in cisplatin tolerance, these all being upregulated in cisplatin-resistant cells. Furthermore, it illustrates the multifactorial nature of cisplatin resistance by examining the diversity of dysregulated pathways present in cisplatin-resistant NSCLC cells based on KEGG pathway analysis.

Evaluation of the relationship between the IL-17A gene expression level and regulatory miRNA-9 in relation to tumor progression in patients with non-small cell lung cancer: a pilot study.

A pro-inflammatory cytokine, IL-17A, is associated with increased risk of developing numerous cancers, including non-small cell lung cancer (NSCLC). IL-17A is a target gene for miR-9. This encouraged us to analyze these two genes in terms of their usefulness as prognostic markers in NSCLC. The expression levels of IL-17A gene and miR-9 was assessed in 26 NSCLC tissue samples and 26 unchanged lung tissue adjacent to lung tumors (control tissue), using qPCR. In both tissue groups, a decreased expression of IL-17A was observed in 100% of samples. Increased expression of miRNA-9 was observed in 92% of tumor samples, and in 100% of control samples. Neither statistical differences in the level of expression IL-17A depending on the patient's age, gender, smoking status, nor histopathology of the cancer was found. Regarding the presence of nodule metastasis ('N' value in TNM classification), significantly lower expression level of IL-17A was observed in cN2 as compared with cN1 group. Additionally, statistically lower IL-17A expression was found in III versus II tumor stage (cAJCC classification). Significant negative correlation between both studied genes was revealed in SCC subgroup. This leads to the conclusion that miRNA-9 can regulate the expression of IL-17A as an IL-17A mRNA antagonistic mediator. Inhibition of proinflammatory action of IL-17A in correlation with tumor progression can be related to various activity of Th17 cells on cancer development according to its immunogenicity, and also may suggest suppressive role of IL-17A in tumor progression. However, because of low number of analyzed samples, further studies on the functional role of IL-17A in development and/or progression NSCLC seem warranted.

Type of training has a significant influence on the GH/IGF-1 axis but not on regulating miRNAs.

The growth hormone (GH)/insulin-like growth factor-1 axis is responsible for glucose homeostasis. In the present study we assessed the expression levels of miRNA-124, miRNA-210 and miRNA-375 and immunoexpression of IGFBP-3 in relation to the concentrations of IGF-1 and glucose in athletes performing different types of effort. Sixty-six young male athletes (age 25.4±4.1 years) were divided into: group EN (33 male athletes; age 25.6±4.4 years) with endurance-type efforts (disciplines: triathlon, long distance running, cycling) and group ST (33 male athletes; age 25.2±3.9 years) with strength-type efforts (disciplines: weightlifting, body building, CrossFit). The control group consisted of 28 non-training men (age 29.1±4.7 years). Statistically significantly higher IGF-1 concentration and lower glucose concentration (P<0.05) in serum were observed in the group of athletes (vs. controls). Immunoexpression of IGFBP-3 was higher in athletes (vs. controls), and a higher value of immunoexpression was obtained in athlete group ST vs. group EN (P>0.05). Levels of expression of miRNA-210 and miRNA-375 were higher in athletes vs. controls (P>0.05). The obtained data confirmed the importance of the somatotropic axis in the regulation of metabolic adaptation to physical exercise. The detected variation in the concentrations and expression levels of the studied molecules involved in the somatotropic axis in athletes confirmed the role of the somatotropic axis in adaptation to physical effort. Statistically significant reduction of glucose concentration and the highest expression of IGF-1in serum in athletes suggest the anabolic effect of IGF-1 through insulin receptors on many tissues under the influence of moderate physical exercises (mainly during resistance training).

Expression of selected miRNA, RARß and FHIT genes in BALf of squamous cell lung cancer (squamous-cell carcinoma, SCC) patients: a pilot study.

Two suppressor genes which often undergo epigenetic silencing during the early stages of lung carcinogenesis are those encoding retinoic acid receptor-ß (RARß) and Fhit protein (FHIT). RARß expression is regulated by miRNA-34a and miRNA-141, and FHIT expression by miRNA-143 and miRNA-217. The aim of the study was to assess how selected miRNAs regulate the expression of their targeted genes in bronchoalveolar lavage fluid (BALf), obtained from patients with SCC of the lung. It also examines the relationship between the genetic findings and the clinical and pathomorphological features of the tumor. A total of 50 BALf samples were taken: 25 from patients with SCC and 25 from healthy donors. The expression (RQ) of the selected genes was analyzed by qPCR, as well as the miRNA level, with a particular emphasis on the relationship between the expression of the genes themselves and their corresponding miRNAs; in addition, the expression of the genes and miRNAs were compared with the pathomorphological features of the tumor and the clinical features of patients. Analysis of the RQ values showed downregulation of RARß, FHIT and miRNA-34a and increased expression of miRNA-141, miRNA-143 and miRNA-217 in all BALf samples (P > 0.05). No correlation was found between the expression of the selected genes and corresponding miRNAs, history of smoking, cancer stage, age and sex of the patients. The presence of the selected genes and miRNAs in BALf material does not seem to have diagnostic potential in patients with SCC; however, the results should be verified on a larger group of patients.

An assessment of the relationship between the expression of CCR7/CCL19 axis and selected regulatory miRNAs in non-small cell lung cancer.

CC chemokine receptor type 7 (CCR7) and its ligands has been implicated in the occurrence and progression of NSCLC. Previous studies have revealed that the diagnostic value of CCR7/CCL19 axis in lung tumorigenesis remains controversial. The present study evaluates the relationship between the mRNA expression of CCR7/CCL19 axis and selected regulatory miRNAs in NSCLC patients. It analyzes the expression level of CCR7 mRNA and its ligand in tumor tissue in relation to expression level of two miRNAs: miR let-7a and miR-335, as transcriptional regulators of study genes. Twenty-seven patients (n = 27) were enrolled. The expression of the studied genes and miRNAs was evaluated by qPCR. Tumour tissue fragments, adjacent macroscopically-unchanged lung tissue (control) and patient serum were used as biological material for study. Elevated expression of CCR7 and CCL19 mRNA was observed in patients with metastasis to lymph nodes. We noticed upregulated miR-335 expression and downregulated miR let-7a expression in patient serum with regard to AJCC tumor staging. Higher miR-335 expression and lower miR let-7a expression level was observed in patients with metastasis to lymph node. The presence of changes observed in the expression level of miR-335 and miR let-7a in the serum of NSCLC patients in relation to lymph node metastases and tumor stage may serve as a non-invasive molecular biomarker of tumor progression; however, this observation requires further investigation.

MicroRNA Profile and Adaptive Response to Exercise Training: A Review.

MicroRNAs are small non-coding regulatory RNAs which may be released into the systemic circulation as a consequence of the body's adaptation to exercise. The expression profile of circulating miRNAs (ci-miRNAs) has been proposed as a potential diagnostic biomarker for adaptive responses of particular systems to physical exertion. Several miRNAs are recognized as regulators of signalling pathways such as the IGF1/PI3K/AKT/mTOR axis, relevant to exercise adaptation. MicroRNA levels may fluctuate depending on training type/exercise regimen in correlation with phenotypic features such as VO2 max. Muscle-specific miRNAs have been proposed as regulators of skeletal muscle/myocardial interactions during physical exertion, thereby facilitating adaptation. Differential expression of miRNAs may relate to molecular patterns of communication triggered during/after exercise as response, recovery and adaptation mechanisms to training load. This review highlights recent findings and the potential significance of specific miRNAs in the process of exercise adaptation. Altered ci-miRNA profiles following exercise suggest that they may be useful biomarkers of health and adaptation to intervention strategies. Identification of the concert of miRNA expression signatures together with their targets is critical towards understanding gene regulation in this context. Understanding how the external environment influences gene expression via miRNAs will provide insight into potential therapeutic target strategies for disease.

The association between airway eosinophilic inflammation and IL-33 in stable non-atopic COPD.

BACKGROUND: Interleukin(IL)-33 is an epithelial alarmin important for eosinophil maturation, activation and survival. The aim of this study was to examine the association between IL-33, its receptor expression and airway eosinophilic inflammation in non-atopic COPD. METHODS: IL-33 concentrations were measured in exhaled breath condensate (EBC) collected from healthy non-smokers, asthmatics and non-atopic COPD subjects using ELISA. Serum and sputum samples were collected from healthy non-smokers, healthy smokers and non-atopic COPD patients. Based on sputum eosinophil count, COPD subjects were divided into subgroups with airway eosinophilic inflammation (sputum eosinophils > 3%) or without (sputum eosinophils ≤3%). IL-33 and soluble form of IL-33 receptor (sST2) protein concentrations were measured in serum and sputum supernatants using ELISA. ST2 mRNA expression was measured in peripheral mononuclear cells and sputum cells by qPCR. Hemopoietic progenitor cells (HPC) expressing ST2 and intracellular IL-5 were enumerated in blood and induced sputum by means of flow cytometry. RESULTS: IL-33 levels in EBC were increased in COPD patients to a similar extent as in asthma and correlated with blood eosinophil count. Furthermore, serum and sputum IL-33 levels were higher in COPD subjects with sputum eosinophilia than in those with a sputum eosinophil count ≤3% (p < 0.001 for both). ST2 mRNA was overexpressed in sputum cells obtained from COPD patients with airway eosinophilic inflammation compared to those without sputum eosinophilia (p < 0.01). Similarly, ST2 + IL-5+ HPC numbers were increased in the sputum of COPD patients with airway eosinophilia (p < 0.001). CONCLUSIONS: Our results indicate that IL-33 is involved in the development of eosinophilic airway inflammation in non-atopic COPD patients.

Expression analysis of selected classes of circulating exosomal miRNAs in soccer players as an indicator of adaptation to physical activity.

Recently studies have shown that, depending on the type of training and its duration, the expression levels of selected circulating myomiRNAs (c-miR-27a,b, c-miR-29a,b,c, c-miR-133a) differ and correlate with the physiological indicators of adaptation to physical activity. To analyse the expression of selected classes of miRNAs in soccer players during different periods of their training cycle. The study involved 22 soccer players aged 17-18 years. The multi-stage 20-m shuttle run test was used to estimate VO2 max among the soccer players. Samples serum were collected at baseline (time point I), after one week (time point II), and after 2 months of training (time point III). The analysis of the relative quantification (RQ) level of three exosomal myomiRNAs, c-miRNA-27b, c-miR-29a, and c-miR-133, was performed by quantitative polymerase chain reaction (qPCR) at three time points - before the training, after 1 week of training and after the completion of two months of competition season training. The expression analysis showed low expression levels (according to references) of all evaluated myomiRNAs before the training cycle. Analysis performed after a week of the training cycle and after completion of the entire training cycle showed elevated expression of all tested myomiRNAs. Statistical analysis revealed significant differences between the first and the second time point in soccer players for c-miR-27b and c-miR-29a; between the first and the third time point for c-miR-27b and c-miR-29a; and between the second and the third time point for c-miR-27b. Statistical analysis showed a positive correlation between the levels of c-miR-29a and VO2 max. Two months of training affected the expression of c-miR-27b and miR-29a in soccer players. The increased expression of c-miR-27b and c-miR-29 with training could indicate their probable role in the adaptation process that takes place in the muscular system. Possibly, the expression of c-miR-29a will be found to be involved in cardiorespiratory fitness in future research.

Altered miRNA expression in pulmonary sarcoidosis.

BACKGROUND: miRNAs control important cellular functions including angiogenesis/angiostasis or fibrosis and reveal altered expression during pathological processes in the lung. METHODS: The aim of the study was to investigate the expression of selected miRNAs (miR-let7f, miR-15b, miR-16, miR-20a, miR-27b, miR-128a, miR-130a, miR-192 miR-221, miR-222) in patients with pulmonary sarcoidosis (n = 94) and controls (n = 50). The expression was assessed by q-PCR in BALF cells and peripheral blood lymphocytes (PB lymphocytes). For statistical analysis, the Kruskal-Wallis test, Mann-Whitney U- test, Neuman-Keuls' multiple comparison test, and Spearman's rank correlation were used. RESULTS: In BALF cells, significantly higher expression of miR-192 and miR-221 and lower expression of miR-15b were found in patients than controls. MiR-27b, miR-192 and miR-221 expression was significantly higher in patients without parenchymal involvement (stages I) than those at stages II-IV. Patients with acute disease demonstrated significantly higher miR-27b, miR-192 and miR-221 expression than those with insidious onset. For PB lymphocytes, patients demonstrated significantly greater miR-15b, miR-27b, miR-192, miR-221 and miR-222 expression, but lower miR-let7f and miR-130a expression, than controls. Stage I patients demonstrated significantly higher miR-16 and miR-15b expression than those in stages II-IV, and patients with the acute form demonstrated higher miR-130a and miR-15b expression. In BALF cells, miR-16 and miR-20a expression was significantly higher in patients with lung volume restriction, and miR-let7f was higher in the PB lymphocytes in patients with obturation. Several correlations were observed between the pattern of miRNA expression, lung function parameters and selected laboratory markers. CONCLUSION: The obtained results suggest that the studied miRNAs play a role in the pathogenesis of sarcoidosis, and that some of them might have negative prognostic value.

Immunoexpression of TGF-ß/Smad and VEGF-A proteins in serum and BAL fluid of sarcoidosis patients.

BACKGROUND: The chronic course of pulmonary sarcoidosis can lead to lung dysfunction due to fibrosis, in which the signalling pathways TGF-ß/Smad and VEGF-A may play a key role. METHODS: We evaluated immunoexpression of TGF-ß1, Smad2, 3, and 7, and VEGF-A in serum and bronchoalveolar lavage (BAL) fluid of patients (n = 57) classified according to the presence of lung parenchymal involvement (radiological stage I vs. II-III), acute vs. insidious onset, lung function test (LFT) results, calcium metabolism parameters, percentage of BAL lymphocytes (BAL-L%), BAL CD4(+)/CD8(+) ratio, age, and gender. Immunoexpression analysis of proteins was performed by ELISA. RESULTS: The immunoexpression of all studied proteins were higher in serum than in BAL fluid of patients (p >0.05). The serum levels of TGF-ß1 (p = 0.03), Smad2 (p = 0.01), and VEGF-A (p = 0.0002) were significantly higher in sarcoidosis patients compared to healthy controls. There were no differences within the sarcoidosis group between patients with vs. without parenchymal involvement, acute vs. insidious onset, or patients with normal vs. abnormal spirometry results. In patients with abnormal spirometry results a negative correlation was found between forced vital capacity (FVC) % predicted value and TGF-ß1 immunoexpression in BAL fluid, and positive correlations were observed between the intensity of lung parenchymal changes estimated by high-resolution computed tomography (HRCT scores) and Smad 2 level in serum. CONCLUSIONS: TGF-ß/Smad signalling pathway and VEGF-A participate in the pathogenesis of sarcoidosis. BAL TGF-ß1, and Smad 2 in serum seem to be promising biomarkers with negative prognostic value, but further studies are required to confirmed our observations.

Quantitative analysis of mRNA expression levels and DNA methylation profiles of three neighboring genes: FUS1, NPRL2/G21 and RASSF1A in non-small cell lung cancer patients.

BACKGROUND: Tumor suppressor gene (TSG) inactivation plays a crucial role in carcinogenesis. FUS1, NPRL2/G21 and RASSF1A are TSGs from LUCA region at 3p21.3, a critical chromosomal region in lung cancer development. The aim of the study was to analyze and compare the expression levels of these 3 TSGs in NSCLC, as well as in macroscopically unchanged lung tissue surrounding the primary lesion, and to look for the possible epigenetic mechanism of TSG inactivation via gene promoter methylation. METHODS: Expression levels of 3 TSGs and 2 DNA methyltransferases, DNMT1 and DNMT3B, were assessed using real-time PCR method (qPCR) in 59 primary non-small cell lung tumors and the matched macroscopically unchanged lung tissue samples. Promoter methylation status of TSGs was analyzed using methylation-specific PCRs (MSP method) and Methylation Index (MI) value was calculated for each gene. RESULTS: The expression of all three TSGs were significantly different between NSCLC subtypes: RASSF1A and FUS1 expression levels were significantly lower in squamous cell carcinoma (SCC), and NPRL2/G21 in adenocarcinoma (AC). RASSF1A showed significantly lower expression in tumors vs macroscopically unchanged lung tissues. Methylation frequency was 38-76%, depending on the gene. The highest MI value was found for RASSF1A (52%) and the lowest for NPRL2/G21 (5%). The simultaneous decreased expression and methylation of at least one RASSF1A allele was observed in 71% tumor samples. Inverse correlation between gene expression and promoter methylation was found for FUS1 (rs = -0.41) in SCC subtype. Expression levels of DNMTs were significantly increased in 75-92% NSCLCs and were significantly higher in tumors than in normal lung tissue. However, no correlation between mRNA expression levels of DNMTs and DNA methylation status of the studied TSGs was found. CONCLUSIONS: The results indicate the potential role of the studied TSGs in the differentiation of NSCLC histopathological subtypes. The significant differences in RASSF1A expression levels between NSCLC and macroscopically unchanged lung tissue highlight its possible diagnostic role in lung cancer in situ recognition. High percentage of lung tumor samples with simultaneous RASSF1A decreased expression and gene promoter methylation indicates its epigenetic silencing. However, DNMT overexpression doesn't seem to be a critical determinate of its promoter hypermethylation.

Selected molecular events in the pathogenesis of sarcoidosis - recent advances.

Sarcoidosis is an orphan inflammatory disorder that can virtually affect any organ or system in the body, although the lungs and lymph nodes are most frequently involved. Sarcoidosis is believed to derive from an interaction between environmental and genetic agents. Many studies emphasize a strong association between certain human leukocyte antigen (HLA) alleles and sarcoidosis susceptibility. Several new insights have allowed the further evaluation of other candidate genes with a potential function in the immunopathogenesis of sarcoidosis. This review summarizes recent advances in the identification of novel molecular markers that may play a role in different stages of disease, such as the acute phase of inflammation, granuloma formation and fibrosis. Furthermore, this article elucidates the role of both TGF-b/Smad and (HIF)-1a-VEGF-ING-4 signaling pathways in the development of sarcoidosis. The potential epigenetic regulation of the processes occurring in sarcoidosis by miRNA is also discussed.