Exosomal miR-1298 and lncRNA-RP11-583F2.2 Expression in Hepato-cellular Carcinoma.

AIM: The aim of this study was to explore the expression of exosomal non-coding RNAs (ncRNAs) in the sera of patients with HCC versus control. METHODS: Firstly, Bioinformatics analysis was conducted to retrieve ncRNAs specific to HCC (hsa-miRNA-1298 and lncRNA-RP11-583F2.2). Afterwards, extraction and characterization of exosomes were performed. We measured the expression of the chosen exosomal RNAs by reverse transcriptase quantitative real-time PCR in sera of 60 patients with HCC, 42 patients with chronic hepatitis C (CHC) infection and 18 healthy normal volunteers. RESULTS: The exosomal ncRNAs [hsa-miRNA-1298, lncRNA-RP11-583F2.2] had better sensitivity and specificity than alpha-fetoprotein (AFP) in HCC diagnosis. CONCLUSION: The exosomal hsa-miRNA-1298, lncRNA-RP11-583F2.2 can be potential biomarkers for HCC diagnosis.

Vitamin D receptor gene methylation in hepatocellular carcinoma.

Worldwide, hepatocellular carcinoma (HCC) is the major subtype of primary liver cancers. HCC is typically diagnosed late in its course. With respect to cancer, the genomic actions of vitamin D are mediated through binding to the Vitamin D Receptor (VDR), which allows it to modulate the expression of genes in a cell-and tissue-specific manner. Epigenetics is a rapidly evolving field of genetic study applicable to HCC. Changes in DNA methylation patterns are thought to be early events in hepatocarcinogenesis. Curcumin has great potential as an epigenetic agent. Accordingly, the current study has been designed to study the methylation status of VDR gene promoter for the first time in HCC aiming to find its clinical significance and potential screening role in chronic Liver Disease (CLD). Additionally, we aimed to investigate, the effect of Curcumin on HCC cell line, aiming to discover new therapeutic targets through epigenetics. This study was conducted on 45 formalin-fixed, paraffin-embedded liver tissue blocks including 15 HCC samples (group A), 15 CLD samples (group B) and 15 apparently normal tissue taken from around benign lesions (group C). Methylation Specific Restriction Digestion and qPCR were done on all samples after DNA extraction. The percentage of VDR gene promoter methylation was significantly higher in the HCC group compared to both CLD and control groups (p < 0.01). VDR promoter methylation by (MS-qPCR) was decreased and the relative expression of VDR by (qRT-PCR) was markedly increased in a dose-dependent fashion in cells grown in Curcumin-adequate medium. In conclusion, this study may open a new gate for the use of VDR promoter methylation as a potential biomarker in HCC.

MicroRNA-181a and its target Smad 7 as potential biomarkers for tracking child acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common pediatric hematologic tumor. MiR-181a was expected to have a role in the development of hematological malignancies; it might act as tumor suppressor or oncogene. Smad7 was selected as miR-181a target pair. It is a negative regulator for the TGF-ß1 signaling pathway. In this study, relative expression levels of miR-181a by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), both Smad 7 and TGF-ß1 proteins levels by enzyme linked immunosorbent assay (ELISA) were all measured in serum of 60 child, 30 with ALL and 30 age and sex matched healthy child as control group. MiR-181a expression showed highly significant decrease; plus a significant increase and decrease of Smad7 and TGF-ß1 protein levels respectively, in serum samples of ALL as compared to control group. MiR-181a expression achieved a highly significant positive and a significant negative correlation with TGF-ß1 and Smad7 respectively. Furthermore, the levels of Smad7 and TGF-ß1 were negatively correlated with each other (p<0.05). Although, positivity rate of both Smad7 and TGF-ß1 in ALL group increased with presence of hepatosplenomegaly, still there was no statistical significance. In conclusion, miR-181a could act as a tumor suppressor in pediatric ALL with over expression of its target pair, Smad7. Smad7 regulates TGF-ß1 signaling via a negative feedback loop and mediates the interaction between TGF-ß1 and other signaling pathways; suggesting that Smad7 over expression may have therapeutic potential in ALL.

Role of microRNA-7 and selenoprotein P in hepatocellular carcinoma.

There is an obvious need to diagnose hepatocellular carcinoma using novel non-invasive and sensitive biomarkers. In this regard, the aim of this study was to evaluate and correlate both relative quantification of microRNA-7 using quantitative real time polymerase chain reaction and quantitative analysis of selenoprotein P using enzyme-linked immunosorbent assay in sera of hepatocellular carcinoma patients, chronic liver disease patients, as well as normal healthy subjects in order to establish a new diagnostic biomarker with a valid non-invasive technique. In addition, this study aimed to investigate whether changes in selenium supply affect microRNA-7 expression and selenoprotein P levels in human hepatocarcinoma cell line (HepG2). The results showed a highly significant decrease in serum microRNA-7 relative quantification values and selenoprotein P levels in malignant group in comparison with benign and control groups. The best cutoff for serum microRNA-7 and selenoprotein P to discriminate hepatocellular carcinoma group from benign and control groups was 0.06 and 4.30 mg/L, respectively. Furthermore, this study showed that changes in selenium supply to HepG2 cell line can alter the microRNA-7 profile and are paralleled by changes in the concentration of its target protein (selenoprotein P). Hence, serum microRNA-7 and selenoprotein P appear to be potential non-invasive diagnostic markers for hepatocellular carcinoma. Moreover, the results suggest that selenium could be used as an anticancer therapy for hepatocellular carcinoma by affecting both microRNA-7 and selenoprotein P.

Evaluation of matrix metalloproteinase-2 in lung cancer.

PURPOSE: There is an obvious need to diagnose lung cancer using novel noninvasive and sensitive biomarkers. In this regard, the aim of the present study was to evaluate and compare sputum matrix metalloproteinase 2 (MMP-2) in relation to serum MMP-2 of lung cancer patients and other nonmalignant lung diseases in order to establish a new diagnostic and prognostic biomarker with a valid noninvasive technique. EXPERIMENTAL DESIGN: Group 1 included 32 newly diagnosed lung cancer patients and group 2 included 20 patients with benign pulmonary diseases. In addition, 38 healthy subjects served as control group. MMP-2 activity levels were evaluated in serum and sputum samples of the studied groups using ELISA and zymography techniques. RESULTS: There was a highly significant increase in serum and sputum MMP-2 levels in malignant group in comparison with benign and control groups. In addition, there was a significant difference in the levels of serum and sputum MMP-2 as regards the different histopathological types of lung cancer and advanced stages of lung cancer. Gelatin zymography was used to confirm the enzymatic activity of MMP-2. A higher MMP-2 activity was detected in lung cancer group in comparison with benign and control groups. CONCLUSIONS AND CLINICAL RELEVANCE: Serum and to a larger extent sputum MMP-2 appear to be potential noninvasive markers for detecting lung cancer.

Oxidant and antioxidant of arylesterase and paraoxonase as biomarkers in patients with hepatitis C virus.

OBJECTIVES: Oxidative stress plays a role in the pathogenesis in patients with HCV infection. The objective of this study was to evaluate oxidant and antioxidant biomarkers in patients with HCV. DESIGN AND METHODS: Serum malonaldehyde (MDA) and nitric oxide (NO) levels and the activities of myeloperoxidase (MPO), arylesterase (AE) and paraoxonase-1 (PON1) were determined in 23 chronic and 21 cirrhotic patients with HCV and 21 healthy subjects. RESULTS: Cirrhotic patients with HCV had higher serum NO level and MPO activity while lower AE and PON1 activities than the chronic. Significant inverse correlation was observed between MDA and PON1 activity in patients with HCV. The most significant HCV biomarker was MDA, AE, NO and PON1. The best combined ones for sensitivity, specificity were MDA+albumin, PON1+AST, and PON1+albumin. CONCLUSIONS: The use of the MDA, MPO, AE, NO and PON1 as biomarkers might be useful tools, helping in the monitoring of patients with HCV.

Detection of telomerase in urine by 3 methods: evaluation of diagnostic accuracy for bladder cancer.

PURPOSE: New, noninvasive methods are needed for the diagnosis, followup and screening of patients with bladder cancer. Three methods of detecting telomerase were evaluated in this aspect. MATERIALS AND METHODS: This study included 200 patients diagnosed with bladder carcinoma, 85 with benign bladder lesions and 30 healthy individuals who served as the control group. All underwent serological schistosomiasis antibody assay in serum, urine cytology and estimation of relative telomerase activity by telomeric repeat amplification protocol, human telomerase RNA by reverse transcriptase-polymerase chain reaction and human telomerase reverse transcriptase by real-time reverse transcriptase-polymerase chain reaction in urothelial cells from voided urine. RESULTS: The concordance between the positive rates of telomerase detected by the 3 methods was high (90% to 95%). Results were significantly higher in the malignant group than in the benign and control groups. There was a significant difference among the results of the 3 methods in relation to different clinicopathological factors. Overall the sensitivity of human telomerase reverse transcriptase for detecting bladder cancer was the highest compared to that of human telomerase RNA, relative telomerase activity and urine cytology (96%, 92%, 75% and 75%, respectively). Combinations of telomerase results with urine cytology were not useful except in cases of relative telomerase activity. CONCLUSIONS: Detection of human telomerase reverse transcriptase in urine by real-time polymerase chain reaction, followed by human telomerase RNA by reverse transcriptase-polymerase chain reaction, improves sensitivity and specificity for the diagnosis of bladder cancer. However, regarding cost-effectiveness, human telomerase RNA is superior.

Noninvasive diagnosis of bladder cancer by detection of matrix metalloproteinases (MMP-2 and MMP-9) and their inhibitor (TIMP-2) in urine.

OBJECTIVES: TIMPs control the activity of MMPs, one of the key molecules for tumor invasion and metastasis. The aim of this study was to assess the usefulness of MMP-2 and MMP-9 in relation to their inhibitor (TIMP2) as noninvasive diagnostic tests for bilharzial bladder cancer. MATERIAL AND METHODS: Voided urine samples were provided from 244 subjects (154 bladder cancer [136 bilharzial]; 60 benign urologic disorders; 30 healthy volunteers). Urine sediment was used for cytology, and the supernatant for estimation of MMPs and TIMP-2 by ELISA and gelatin zymography. RESULTS: The best cut-off values for the investigated markers were determined by ROC curve. Positivity rates and median levels for MMP-2, MMP-9, TIMP-2, MMP-2/TIMP-2, and MMP-9/TIMP-2 showed significant difference among the three investigated groups (p<0.001). MMP-9 and MMP-2/TIMP-2 were related to pathologic type, MMP-2/TIMP-2 was inversely related to the grade, and MMP-9/TIMP-2 was related to bilharziasis (p<0.05). MMP zymography results were comparable to those from ELISA. CONCLUSION: The sensitivity and specificity of MMP zymography, MMP-9/TIMP-2 ratio, and MMP-2/TIMP2 ratio were superior among all investigated parameters; furthermore, combined testing of cytology with them improves the sensitivity even in superficial and low-grade tumors.

Prognostic Significance of Ets-1 and the Matrix Metalloproteinase-2 to Tissue Inhibitor of Matrix Metalloproteinase-2 Ratio in Egyptian Epithelial Ovarian Cancer Patients.

The invasive potential of epithelial ovarian cancer is the main factor determining its biological behavior. Ets-1 transcription factor is involved in the activation of several proteases participating in tumor invasion and metastasis. The balance of matrix metalloproteinase-2 and its inhibitor is important in the regulation of tumor invasion. This study included 31 tumors from patients with epithelial ovarian cancer in different stages, and 20 tissue samples from benign ovarian lesions as a control group. Ets-1 was assessed using immunohistochemistry. Matrix metalloproteinase-2 (MMP-2) and the MMP-2 inhibitor (TIMP-2) were measured in the cytosolic fractions using enzyme immunoassay. MMP-2 results were confirmed by gelatin zymography. ETS-1 was expressed only in the malignant group. MMP-2 and the MMP-2:TIMP-2 ratio were significantly higher in the malignant group (p=0.045 and 0.026, respectively) with significant correlation to stage and poor survival above the specified cut-off values (p=0.006 and 0.002, respectively). Log rank of Kaplan-Meier survival analysis was significant for FIGO stage, MMP-2 and the MMP-2:TIMP-2 ratio (p<0.05). Multivariate analysis demonstrated that the MMP-2:TIMP-2 ratio is an independent prognostic parameter. Ets-1 could be a future target for therapeutic strategies. Moreover, the MMP-2:TIMP-2 ratio might serve as a new independent prognostic indicator of poor prognosis in epithelial ovarian cancer patients.

Detection of urokinase plasminogen activator receptor and c-erbB-2 in sera of patients with breast and ovarian carcinoma.

The role of urokinase plasminogen activator receptor (uPAR) and c-erbB-2 in breast and ovarian cancer was investigated. Eighty patients of breast and ovarian cancer and benign lesions, as well as twenty normal controls were evaluated for the expression of c-erbB-2 by Western blotting and uPAR levels by ELISA. The c-erbB-2 and uPAR showed a significant increase in both types of cancer investigated compared to normal control and benign lesions. The frequency of c-erbB-2 was significantly higher in breast cancer lesions (p < 0.01). Levels of CA15.3 in breast cancer and CA125 in ovarian cancer were significantly higher in cases expressing c-erbB-2 (p < 0.01) than in negative c-erbB-2 cases. The uPAR showed a significant positive correlation with advanced stages of breast cancer (r = 0.7971) and ovarian cancer (r = 0.83662), while significant correlations were found for CA15.3 in breast cancer (r = 0.64967) and CA125 in ovarian cancer (r = 0.83996). Taken together, our data suggest that the c-erbB-2 and uPAR in the sera of ovarian and breast cancer act as valuable markers for the evaluation of the patients preoperatively.