Investigation of miR221 and miR222 as Biomarkers in Non-small Cell Lung Cancer.

BACKGROUND/AIM: MicroRNAs (miRNA) are a class of small non-coding RNAs of 18-25 nucleotides, which regulate gene expression at the post-transcriptional level by disrupting or blocking translation of messenger RNA targets. Non-small cell lung cancer (NSCLC) represents approximately 85% of all lung cancers. Early and accurate diagnosis of the disease affects the probability of success of treatment. The purpose of this study was to investigate the expression levels of serum specific miRNA221 and miRNA222 as a biomarker in NSCLC. MATERIALS AND METHODS: Thirty-two NSCLC cases and 30 healthy control cases that were diagnosed at Istanbul Yedikule Chest Diseases and Thoracic Surgery Training Hospital were included in this study. miRNAs were detected using miRNA-specific quantitative real-time-PCR. The relative expression of miRNAs was calculated using the 2-ΔΔCt method. RESULTS: miR221 and miR222 showed 1.46 and 1.63-fold higher expression in the samples from patients with NSCLC compared to controls, and the difference of expression was statistically significant for miR221 (p=0.000095) but not for miR222 (p=0.084470). In the presence of metastasis in NSCLC patients, miR221 levels were 2.33-fold higher compared to non-metastatic cases (p=0.014), and those of miR221 and miR222 were expressed 1.44 and 1.52-fold higher, respectively, in advanced stage compared to early stage (p=0.000387, p=0.000302). CONCLUSION: The levels of miR221 and miR222 in the serum of patients could be used as biomarkers for the diagnosis, treatment, and prognosis of NSCLC.

Increased serum soluble lectin-like oxidized low-density lipoprotein receptor-1 levels in patients with biopsy-proven nonalcoholic fatty liver disease.

AIM: To analyze the relationship between the serum lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) levels and clinical and histopathological features of biopsy-confirmed nonalcoholic fatty liver disease (NAFLD) patients. METHODS: Fifty-three consecutive, biopsy-proven NAFLD patients (31 males and 22 females, mean age 42.5 ± 9.6 years) and 26 age- and gender-matched, healthy controls (14 males and 12 females, mean age 39 ± 10.7 years) were included. The patients with NAFLD were consecutive patients who had been admitted to the hepatology outpatient clinic within the last year and had been diagnosed with NAFLD as the result of liver biopsy. The healthy controls were individuals who attended the outpatient clinic for routine health control and had no known chronic illnesses. The histological evaluation was conducted according to the NAFLD activity scoring system recommended by The National Institute of Diabetes and Digestive and Kidney Diseases Nonalcoholic Steatohepatitis Clinical Research Network. The serum LOX-1 levels were measured using an ELISA kit (Life Science Inc. USCN. Wuhan, Catalog No. E1859Hu) in both patients and healthy controls. A receiver operating characteristic (ROC) curve analysis was used to identify the optimal cutoff value of LOX-1 and thereby distinguish between patients with nonalcoholic steatohepatitis (NASH) and healthy controls. A P-value < 0.05 was considered statistically significant. RESULTS: NAFLD and healthy control groups were similar in terms of age and sex. NAFLD patients consisted of 8 patients with simple steatosis (15%), 27 with borderline NASH (51%) and 18 with definitive NASH (34%). Metabolic syndrome was found in 62.2% of the patients with NAFLD. The mean serum LOX-1 level in biopsy-proven NAFLD patients was 8.49 ± 6.43 ng/mL compared to 4.08 ± 4.32 ng/mL in healthy controls (P = 0.001). The LOX-1 levels were significantly different between controls, simple steatosis and NASH (borderline+definite) cases (4.08 ± 4.32 ng/mL, 6.1 ± 6.16 ng/mL, 8.92 ± 6.45 ng/mL, respectively, P = 0.004). When the cut-off value for the serum LOX-1 level was set at 5.35 ng/mL, and a ROC curve analysis was performed to distinguish between steatohepatitis patients and controls; the sensitivity and specificity of the serum LOX-1 level were 69.8% and 69.2%, respectively. CONCLUSION: The serum LOX-1 levels were significantly higher in NAFLD patients than in healthy controls. Additionally, the serum LOX-1 levels could differentiate between steatohepatitis patients and healthy controls.

The assessment of the relationship between variations in the apelin gene and coronary artery disease in Turkish population.

OBJECTIVE: Apelin is a novel endogenous peptide with inotropic and vasodilatory properties and is the ligand for the angiotensin receptor-like 1 (APJ) receptor. The aim of the study was to investigate the association of 2 single-nucleotide polymorphisms (SNPs) in the apelin gene with susceptibility to coronary artery disease (CAD) in the Turkish population. METHODS: The present observational case-control study consisted of 244 subjects (134 angiographically proven CAD patients and 110 healthy controls) aged 30-65 years. The association of 2 SNPs (rs3115758 and rs3115759) in the apelin gene and CAD risk was investigated. Real-time polymerase chain reaction (RT-PCR) was used to analyze the 2 SNPs in both the CAD and the healthy subjects. Allele and genotype frequencies between patients and control groups were compared using the Chi-square (χ2) test. The relationships of the 2 polymorphisms with the presence of CAD were determined with multiple binary logistic regression analysis after adjustment for CAD risk factors. RESULTS: TT and AA risk genotypes of the rs3115758 and rs3115759 variants in the apelin gene were found to be significantly related with the risk of CAD with the same power (OR: 6.36, 95% CI: 1.41-28.6) (p=0.007). After adjustments for traditional CAD risk factors, the homozygous TT genotype for rs3115758 and AA genotype for rs3115759 increased the CAD risk, both with an OR of 5.91. CONCLUSION: Genetic variants in the apelin gene are significantly associated with the risk of CAD in the Turkish population.

Investigation of the association of hRRM1 and p53R2 gene polymorphisms in head and neck squamous cell carcinomas.

Head and neck squamous epithelial cell cancer (HNSCC), the world's fifth most common type of cancers, is associated with short life expectancy and high death rates if not detected in early stages. The aim of this study was to investigate hRRM1 and p53R2 gene polymorphisms by using real-time PCR technique in patients with head and neck cancer. In total, 87 patients with head and neck malignancies and 87 control group who have not any malignancies were included in the study between January 2011 and February 2012 in Istanbul University Faculty of Medicine Department of ORL. In the study, real-time PCR was used to detect hRRM1 (rs12806698 C/A) and p53R2 (rs2290707 G/T) gene polymorphisms in Turkish HNSCC patients and healthy individuals. Genomic DNA isolation was performed according to the kit protocol with spin column. LightCycler 1.5 system was used to perform SNP genotyping using hybridization probes consisting of 3'-fluorescein and a 5'-LightCycler Red labeled pair of oligonucleotide probes. There were significant differences in the distribution of hRRM1 genotypes. Frequency of individuals with hRRM1 AA genotype was higher in patients with less differentiation when compared with well differentiation [p 0.025, Fisher's exact test, odds ratio (OR) 0.140, 95 % confidence intervals (CI) 0.024-0.797]. It is observed that A allele carriers have nearly twofold risk for development of the disease (p = 0.022; χ (2) 5.24; OR 2.02, 95 % CI 1.10-3.72).

GSTT1 is deregulated in left colon tumors.

Our aim was to determine GSTT1 expression levels in left colon tumors and paired normal tissue in order to identify specific alterations in GSTT1 mRNA levels. Alterations in GSTT1 expression in twenty-four left- sided colon tumors and paired cancer free tissue were determined by qRT-PCR. Significant fold changes were determined with t-test. When compared with cancer free tissue, left colon cancers showed a significant decrease in GSTT1 expression. However, GSTT1 mRNA levels among different grades increased gradually in correlation with tumor grade. Our results suggest that downregulation of GSTT1 in left-sided colon cancers is an early event and is reversed with cancer progression, probably due to cellular defense mechanisms as a response to changes in the microenvironment.

The significance of HSP90AA1, HSP90AB1 and HSP90B1 gene polymorphisms in a Turkish population with non-small cell lung cancer.

BACKGROUND/AIM: Heat-shock proteins (HSPs) are molecular chaperones which modify the structures and interactions of other proteins. The aim of our study was to investigate HSP90AA1, HSP90AB1 and HSP90B1 gene polymorphisms in patients with non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: Ninety-seven patients with NSCLC and 97 healthy controls were included in the study. Real-time polymerase chain reaction was used for genotyping. RESULTS: The frequency of mutant CC genotype for HSP90AA1 (rs4947C/T), mutant AA genotype for HSP90AB1 (rs13296A/G) and mutant CC genotype for HSP90B1 (rs2070908 C/G) was significantly higher in the patient group than in controls (p=0.019, p=0.004 and p=0.036, respectively). The frequency of patients with homozygote mutant allele was also significantly higher than that of controls and possessing of the mutant genotype increased the risk for disease by approximately 2.9, 4.8, 1.9 for HSP90AA1, HSP90AB1 and HSP90B1, respectively. The present study appears to be the first of its kind to report data on these gene polymorphisms in patients with NSCLC in the Turkish population.

Investigation of biomarker in laryngeal carcinomas.

BACKGROUND: The aim of the study is to determine whether there is a role of podoplanin and glutathione S-transferases T1 (GST-T1) expression in laryngeal squamous cell carcinoma. METHODS: In this study, 33 patients were enrolled and gene expression analysis was performed by qRT-PCR. The podoplanin and GST-T1 expression patterns were analyzed to determine their correlation with clinicopathologic parameters of laryngeal cancer. RESULTS: Of all included patients, 20 had supraglottic, and 13 had glottic laryngeal cancer. Increased expression of podoplanin was found in seven (35%) supraglottic tumor tissues and seven (53.8%) glottic tumor tissues, but GST-T1 expression was not detected. CONCLUSION: Podoplanin expression did not show any prediction for tumor differentiation, regional metastasis, thyroid cartilage invasion, lymphatic vessel invasion, or tumor differentiation for laryngeal cancer, and also there were no significant differences in podoplanin expression between glottic and supraglottic regions, but extracapsullar extension is almost statistically significance (P = 0.05).

Serum osteopontin levels as a predictor of portal inflammation in patients with nonalcoholic fatty liver disease.

BACKGROUND: Osteopontin is a secreted phosphorylated glycoprotein that is expressed by a variety of cell types and that mediates numerous and diverse biological functions. Osteopontin knockout mice are protected from obesity-induced hepatic steatosis. In the present study, we sought to investigate whether serum osteopontin concentrations are associated with liver histology in patients with nonalcoholic fatty liver disease. METHODS: Serum levels of osteopontin were measured by enzyme-linked immunosorbent assay in 179 well-characterized patients with nonalcoholic fatty liver referred for liver histology and 123 control subjects. RESULTS: Serum osteopontin levels were markedly higher in patients with nonalcoholic fatty liver disease than in controls (p<0.001). Multivariable analysis showed that osteopontin levels were strongly and independently associated with both portal inflammation (ß=0.294, p<0.01) and serum aminotransferase levels (aspartate aminotransferase: ß=0.295, p<0.01; alanine aminotransferase; ß=0.285, p<0.01). CONCLUSION: In summary, these data demonstrate that serum levels of osteopontin are elevated in nonalcoholic fatty liver disease and are a significant independent predictor of portal inflammation in this clinical entity.

Concentrations of connective tissue growth factor in patients with nonalcoholic fatty liver disease: association with liver fibrosis.

AIM: In this study, we aimed to investigate the relationship between the histological fibrosis stage of nonalcoholic fatty liver disease (NAFLD) and serum connective tissue growth factor (CTGF) to determine the usefulness of this relationship in clinical practice. METHODS: Serum samples were collected from 51 patients with biopsy-proven NAFLD and 28 healthy controls, and serum levels of CTGF were assayed by ELISA. RESULTS: Levels of CTGF were significantly higher in patients with NAFLD compared with controls (P=0.001). The serum CTGF levels were significantly increased, that correlated with histological fibrosis stage, in patients with NAFLD [in patients with no fibrosis (stage 0) 308.2 ± 142.9, with mild fibrosis (stage 1-2) 519.9 ± 375.2 and with advanced fibrosis (stage 3-4) 1353.2 ± 610 ng/l, P < 0.001]. Also serum level of CTGF was found as an independent predictor of histological fibrosis stage in patients with NAFLD (ß = 0.662, t=5.6, P <0.001). The area under the ROC curve was estimated 0.931 to separate patients with severe fibrosis from patients with other fibrotic stages. CONCLUSION: Serum levels of CTGF may be a clinical utility for distinguishing NAFLD patients with and without advanced fibrosis.

Analysis of serum micro-RNAs as potential biomarker in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) as a complex disease with genetic and environmental compound is one of the leading causes of death in worldwide. This disease is characterized by lower airway inflammation, and increases risk of lung cancer in smokers. Micro-RNA (miRNA) molecules are key regulators in gene expression that have been widely associated with a several diseases. Differential expression of miRNAs is involved in lung tissue of COPD, but there is no information about biomarker potential of circulating miRNAs in patients. To analyze the miRNA expression profile in COPD, levels of serum miRNAs were profiled by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) array system. The authors examined 72 miRNAs by qRT-PCR array, in 20 COPD patients and 12 control subjects. U6snRNA was used for normalization of the expression of miRNAs for each sample. According to the results, 5 miRNAs were found to be significantly dysregulated. There was down-regulation of miR-20a, miR-28-3p, miR-34c-5p, and miR-100, and up-regulation of miR-7, compared with the controls. This was the first study in COPD for screening of serum miRNAs for searching for biomarker. These results are preliminary screening data and should be confirmed with large patient groups. If so, these miRNAs are likely being involved in pathogenesis of COPD and may give clues for designing therapeutic strategy.

Is MMP-7 gene polymorphism a possible risk factor for chronic obstructive pulmonary disease in Turkish patients.

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease that leads to fixed narrowing of small airways and alveolar wall destruction (emphysema). This study was performed to test the association between MMP-7 (rs155668818) and MMP-12 (rs56184183) polymorphisms in the MMP-7 gene and COPD risk and its severity in the Turkish population. MMP-7 and MMP-12 polymorphisms were genotyped in 85 patients with COPD and 73 healthy control subjects using real-time polymerase chain reaction analysis. There were significant differences in the distribution of MMP-7 genotypes but not in the frequencies of these alleles between COPD patients and controls (p=0.009, p=0.102, respectively). The MMP-7 AA genotype was found to be associated with an increased risk of COPD (p=0.004; odds ratio: 2.576; confidence interval: 1.297-5.119). The lowest values of forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC), FEV1/FVC in patients with GG homozygosity were determined and these values were statistically significant compared to the control subjects (p<0.001, p<0.001, p<0.001). When the present study groups were analyzed for MMP-12 polymorphism, it was found that all the subjects had wild-type genotype for this polymorphism. These findings have suggested that MMP-7 polymorphism might be associated with the risk and progression of COPD in the Turkish population.

Investigation of a possible relationship between EPHX1 gene polymorphisms and colorectal cancer in Turkish society.

Metabolism of chemical carcinogens, including their activation and detoxification, plays a key role in carcinogenesis. Microsomal epoxide hydrolase (EPHX1) has an important role in the metabolism of polycyclic aromatic hydrocarbons and detoxification of procarcinogens. The aim of this study was to investigate the association between colorectal cancer (CRC) development and EPHX1 gene polymorphisms. We investigated the polymorphisms in exon 3 (T>C, Tyr113His) and exon 4 (A>G, His139Arg) of the EPHX1 gene in 68 CRC patients and 116 controls by polymerase chain reaction-restriction fragment length polymorphism. The frequencies of the Try113Try, Try113His, and His113His for EPHX1 exon 3 were 37.9%, 55.2%, and 6.9% in controls and 39.7%, 42.6%, and 17.6% in CRC patients, respectively. Frequencies of EPHX1 exon 4 genotypes were 62.1% His139His, 37.9% His139Arg, and 0% Arg139Arg in the control group and 76.5% His139His, 22.1% His139Arg, and 1.5% Arg139Arg in the patient group. Individuals carrying the EPHX1 exon 3 His113His genotype had a 2.5-fold increased risk (p=0.024), and those carrying the EPHX1 exon 4 His139Arg genotype had decreased risk of CRC compared with controls (p=0.019). Even though exon 3 Tyr113His and exon 4 His139Arg polymorphisms for EPHX1 gene appear to be important factors for CRC risk, further investigations with larger study groups are needed to fully elucidate the role of these polymorphisms in the development of CRC.

Association of serum lipoprotein-associated phospholipase A2 level with nonalcoholic fatty liver disease.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome and is one of the most common causes of chronic liver disease, worldwide. Lipoprotein-associated phospholipase A2 (Lp-PLA2) was recently characterized as a novel inflammatory biomarker that is correlated with several components constituting the metabolic syndrome. METHODS: In this study, we determined the serum levels of Lp-PLA2 in patients with definite nonalcoholic steatohepatitis (NASH, n=25), borderline NASH (n=22), simple fatty liver (n=10), and healthy controls without evidence of liver disease (n=38). The levels of Lp-PLA2 were measured by enzyme-linked immunosorbent assay and compared in the four study groups. Moreover, concentrations of Lp-PLA2 were assessed in relation to the general characteristics of the study participants and the results of liver biopsy. RESULTS: Concentrations of Lp-PLA2 were significantly higher in patients with definite NASH (161.8±0.9 µg/L, P<0.001), borderline NASH (135.4±47.7 µg/L, P=0.001), and simple fatty liver (132.4±46.2 µg/L, P=0.042) compared with healthy controls (86.2±40.7 µg/L). Furthermore, the serum Lp-PLA2 level was strongly associated to histological steatosis scores in patients with NAFLD (ß=0.32, t=2.50, P=0.016). CONCLUSION: Although subject to future confirmation, our data suggest that Lp-PLA2 levels are elevated in NAFLD.