Evaluation of selected IL6/STAT3 pathway molecules and miRNA expression in chronic obstructive pulmonary disease.

COPD has been regarded as a global epidemic due to an increase in pollution and tobacco exposure. Therefore, the study of molecular mechanism as the basis for modern therapy is important. The aim of the study was the assessment of gene expression levels, IL-6, IL-6ST, PIAS3, STAT3, and miRNAs, miRNA-1, miRNA-106b, miRNA-155, in patients with COPD. Induced sputum as well as PBMC were collected from 40 patients clinically verified according to the GOLD 2021 (A-D) classification and from the control group (n = 20). The levels of gene and miRNA expression were analysed by qPCR. In induced sputum IL6 was significantly down-regulated in COPD group compared with control (p = 0.0008), while IL6ST were up-regulated (p = 0.05). The results were also statistically significant for STAT3 (p = 0.04) and miRNA-155 (p = 0.03) with higher expression in the current smokers compared to ex-smokers. Higher expression levels for IL6ST (p = 0.03) in COPD patients with the exacerbation history compared to COPD patients without the exacerbation history were noted. Compared induced sputum and PB lymphocytes we observed higher expression of IL6 (p = 0.0003), STAT3 (p = 0.000001) miRNA-106b (p = 0.000069 and miRNA-155 (p = 0.000016) in induced sputum with lower expression of PIAS3 (p = 0.006), IL6ST (p = 0.002) and miRNA-1 (p = 0.001). Differences in gene expression levels of the IL-6/IL6ST/STAT3 pathway and miRNA depending on the smoking status and classification of patients according to GOLD suggest the importance of these genes in the pathogenesis of COPD and may indicate their potential utility in monitoring the course of the disease.

Expression analysis of three miRNAs, miR-26a, miR-29b and miR-519d, in relation to MMP-2 expression level in non-small cell lung cancer patients: a pilot study.

Lung cancer is the most common cause of death in men and second only to breast cancer in women. MicroRNAs (miRNAs) are involved in tumorigenesis and function as oncogenes or tumor suppressor genes. Among other genes, miRNAs regulate matrix metalloproteinases (MMPs), the proteolytic enzymes playing a significant role in the degradation of extracellular matrix, enhancing tumor invasion and metastasis. The aim of the study was to evaluate the expression levels of selected miRNAs: miR-26a, miR-29b and miR-519d, and their target gene, matrix metalloproteinase-2 (MMP-2) in patients with non-small cell lung cancer (NSCLC). The results were correlated with tumor staging, NSCLC histopathological subtypes and patients' demographical features to assess the possible diagnostic/prognostic value of the studied miRNAs and MMP-2. Total RNA was isolated from 38 NSCLC tissue samples, and the expression analysis was performed using TaqMan(®) probes in qPCR assay. The results indicated underexpression of selected miRNAs and overexpression of MMP-2. The decrease in miRNA-29b expression was statistically significant and differentiated NSCLC histopathological subtypes. Additionally, statistically significant negative correlation was found between MMP-2 expression and its regulatory miR-26a. There are very few studies reporting miRNA-MMPs analysis on mRNA level in lung cancer, and no similar reports are available from Polish population. The results of our pilot study indicated the diagnostic potential of miR-29b and MMP-2, an inverse association between miR-26a and MMP-2, and proved the role of MMP-2 and the studied miRNAs in lung carcinogenesis. Further studies are needed to verify their potential usefulness for the treatment of lung cancer.

RARß Promoter Methylation as an Epigenetic Mechanism of Gene Silencing in Non-small Cell Lung Cancer.

The retinoid acid receptor-p (RARß) gene is one of the tumor suppressor genes (TSGs), which is frequently deleted or epigenetically silenced at an early stage of tumor progression. In this study we investigated the promoter methylation and expression status of the RARß gene in 60 surgically resected non-small cell lung cancer (NSCLC) tissue samples and 60 corresponding unchanged lung tissue samples, using methylation-specific PCR and real-time-polymerase chain reaction (qPCR) techniques. We correlated the results with the pathological features of tumors and clinical characteristics of patients. qPCR analysis detected a significantly lower RARß expression in the patients with adenocarcinoma (AC) and large cell carcinoma (LCC) than in those with squamous cell carcinoma (SCC) (AC vs. SCC, p = 0.032; AC and LCC vs. SCC, p = 0.0 13). Additionally, significantly lower expression of the RARß gene was revealed in the patients with non-squamous cell cancer with a history of smoking assessed as pack-years (PY < 40 vs. PY ≥ 40, p = 0.045). Regarding RARß promoter methylation, we found significant differences in the methylation index in the SCC group when considering pTNM staging; with higher index values in T1a + T1b compared with T2a + T2b and T3 + T4 groups (p = 0.024). There was no correlation between the methylation status and expression level of the RARß gene, which suggests that other molecular mechanisms influence the RARß expression in NSCLC patients. In conclusion, different expression of the RARß gene in SCC and NSCC makes the RARß gene a valuable diagnostic marker for differentiating the NSCLC subtypes.

Expression of HIF-1A/VEGF/ING-4 Axis in Pulmonary Sarcoidosis.

Angiogenesis/angiostasis regulated by hypoxia inducible factor-1A (HIF-1A)/vascular endothelial growth factor (VEGF)/inhibitor of growth protein 4 (ING-4) axis may be crucial for the course and outcome of sarcoidosis. Overexpression of angiogenic factors (activation of VEGF through HIF-1A) may predispose to chronic course and lung fibrosis, whereas immunoangiostasis (related to an overexpression of inhibitory ING-4) may be involved in granuloma formation in early sarcoid inflammation, or sustained or recurrent formation of granulomas. In this work we investigated gene expression of HIF-1A, VEGF and ING-4 in bronchoalveolar fluid (BALF) cells and in peripheral blood (PB) lymphocytes of sarcoidosis patients (n=94), to better understand mechanisms of the disease and to search for its biomarkers. The relative gene expression level (RQ value) was analyzed by qPCR. The results were evaluated according to the presence of lung parenchymal involvement (radiological stage I vs. II-IV), acute vs. insidious onset, lung function tests, calcium metabolism parameters, percentage of lymphocytes (BALL%) and BAL CD4+/CD8+ in BALF, age, and gender. In BALF cells, the ING-4 and VEGF RQ values were increased, while HIF-1A expression was decreased. In PB lymphocytes all studied genes were overexpressed. Higher expression of HIF-1A in PB lymphocytes of patients with abnormal spirometry, and in BALF cells of patients with lung volume restriction was found. VEGF gene expression in BALF cells was also higher in patients with abnormal spirometry. These findings were in line with previous data on the role of HIF-1A/VEGF/ING-4 axis in the pathogenesis of sarcoidosis. Up-regulated HIF-1A and VEGF genes are linked to acknowledged negative prognostics.

TGF-ß and SMADs mRNA Expression in Pulmonary Sarcoidosis.

Lung fibrosis is a complication of sarcoidosis, in which TGF-ß/Smad pathway may play an important role. We evaluated gene expression of TGF-ß1, SMAD2, 3 and 7 in bronchoalveolar lavage (BAL) cells and peripheral blood (PB) lymphocytes of sarcoidosis patients (n=94) to better understand the mechanisms of sarcoid inflammation. The relative gene expression was analyzed by qPCR method. Selected clinical/radiological features and biochemical markers were taken into account in the analysis. We found that TGF-ß1 and SMAD3 expressions in PB lymphocytes were significantly higher in sarcoidosis patients. Up-regulation of SMAD7 (inhibitory Smad) and down-regulation of SMAD3 in BAL cells in all subgroups were found. The expression of TGF-ß1 in PB lymphocytes was the highest in patients with lung parenchymal involvement and in the insidious onset phenotype. The expression of TGF-ß1 in BAL cells was higher in patients with abnormal spirometry (p=0.012), and TGF-ß1 and SMAD3 in patients with restrictive pattern (p=0.034 and 0.031, respectively). Several statistically significant negative correlations were found between the expression levels of SMAD2 and 3 in BAL cells and various LFT parameters. We conclude that TGF-ß/Smad pathway is involved in the pathogenesis of pulmonary sarcoidosis. These biomarkers (especially TGF-ß1, SMAD2 and 3) are of a negative prognostic value.

Decreased FAM107A Expression in Patients with Non-small Cell Lung Cancer.

Lung cancer is the leading cause of cancer-related death in the world. Early detection, based on molecular markers, could decrease mortality from this disease. Tumor development is often associated with inactivation or loss of tumor suppressor genes (TSGs). The aim of the present study was to analyze the expression level of FAM107A gene, a TSG located in 3p21.1, in lung cancer tumors and in tumor adjacent normal lung samples. Promoter methylation status of FAM107A was evaluated as the potential mechanism of its epigenetic silencing. The relationship between gene mRNA expression and tumor staging, metastasis status, and non-small cell lung cancer (NSCLC) histopathological subtypes in 60 patients was analyzed. Total RNA was isolated from tissue samples and gene expression was assessed in qPCR assay. Gene promoter methylation status was evaluated in MSP reactions, using bisulfite converted DNA and two pairs of primers: methylated and unmethylated. We found that the expression of the gene was dramatically decreased in all NSCLC samples and was significantly lower than in tumor adjacent normal lung tissue. Promoter methylation of FAM107A gene was confirmed only in the minority of NSCLCs. The results highlight the importance of FAM107A in lung carcinogenesis, although indicate other than promoter hypermethylation mechanism of the gene decreased expression.