Serum MicroRNA profiles in chronic hepatitis C Egyptian patients before and after combined sofosbuvir and daclatasvir treatment.

BACKGROUND: MicroRNAs (miR) are small sequence of nucleotides that can affect multiple genes involved in the hepatitis C virus (HCV) life cycle and disease development. The purpose of the present study was to investigate the clinical significance of serum microRNA profiles in a cohort of Egyptian patients with chronic HCV infection before and after combined sofosbuvir and daclatasvir treatment, as well as to gain a better understanding of the exact interaction mechanism in HCV transcriptional activity via differentially expressed miRNAs. For 12 weeks, 50 patients were eligible for and received sofosbuvir (400 mg daily) and daclatasvir (60 mg daily) treatment. Each patient's blood was obtained twice: once before therapy began and again three months afterwards. RESULTS: The current study found that serum levels of circulating miR-122, miR-221, miR-23a, miR-125, miR-217, miR-224, and miR-181a were high in HCV pre-treatment patients, but after 12 weeks of direct-acting antiviral (DAAs) treatment, there was a statistically significant reduction in expression levels of miR-122, miR-221, miR-23a, miR-125, miR-217, and miR-224 (p < 0.001). There is no statistical significance for miR-181a. CONCLUSION: The key differentially expressed microRNAs before and after the direct-acting antiviral (DAA) regimen were connected to the dynamics of chronic HCV infection, suggesting their potential as predictive biomarkers for HCV clearance after sofosbuvir and daclatasvir therapy.

Clinical and diagnostic characteristics of non-alcoholic fatty liver disease among Egyptian children and adolescents with type1 diabetes.

BACKGROUND: Type 1 diabetes mellitus (T1DM) patients are at an increased risk for non-alcoholic fatty liver disease (NAFLD). This study aimed to evaluate the clinical criteria associated with the diagnosis of Non-Alcoholic Fatty Liver Disease (NAFLD) among T1DM Egyptian children and adolescents. METHODS: 74 T1DM patients aged 8-18 year were enrolled in this cross sectional study. Assessments of Clinical status, anthropometric measures, lipid profile, glycated haemoglobin (HbA1c) and liver enzymes were done. Abdominal Ultrasound evaluation of hepatic steatosis was done. Accordingly, patients were divided into two groups (NAFLD and normal liver group) and compared together. Assessment of liver fibrosis using acoustic radiation force impulse elastography (ARFI) was done. Statistical analysis included; independent t-test, Chi square and Fisher's Exact, Pearson and Spearman tests and Logistic regression models for factors associated with fatty liver were used when appropriate. RESULTS: In this study; 74 patients were enrolled; 37 males (50%) and 37 females with mean age 14.3 ± 3.0 year. The mean insulin dose was 1.1 ± 0.4 U/kg and mean disease duration was 6.3 ± 3.0 year. NAFLD was detected in 46 cases while 28 cases had normal liver as diagnosed by abdominal ultrasound. Cases with NAFLD had statistically significant higher BMI-Z scores, waist/hip, waist/height and sum of skin fold thicknesses compared to those with normal liver (P < 0.05). The mean value of HbA1c % was significantly higher in NAFLD group (P = 0.003). Total cholesterol, triglycerides and LDL serum levels were significantly elevated (p < 0.05), while the HDL level was significantly lower in NAFLD cases (p = 0.001). Although, serum levels of liver enzymes; ALT and AST were significantly higher among cases with NAFLD than in normal liver group (p < 0.05), their means were within normal. Using the ARFI elastography; NAFLD cases exhibited significant fibrosis (F2, 3 and 4). BMI, patient age and female gender were among risk factors for NAFLD. CONCLUSIONS: NAFLD represents a serious consequence in type 1 diabetic children and adolescents that deserves attention especially with poor glycemic control. NAFLD has the potential to evolve to fibrosis. This study demonstrated a very high prevalence of NAFLD in T1D children and adolescents using US which was (62.2%) with the percent of liver fibrosis among the NAFLD cases (F2-F4) using ARFI elastography was 26%. BMI, age of patients and female gender were detected as risk factors for NAFLD.

Detection of DNA methylated microRNAs in hepatocellular carcinoma.

Irregular methylation, including DNA hypomethylation and/or promoter gene CpG hypermethylation, is involved in the pathogenesis of several solid tumors, including liver cancer. miRNAs are small, endogenous, noncoding RNAs that serve as posttranscriptional regulators of gene expression. Previous research has focused on identifying the factors that regulate the expression of miRNAs in hepatic carcinogenesis. The factors that regulate miRNA expression are not clear; in particular, the role of DNA methylation, an epigenetic regulatory factor that controls miRNA transcription, has not been clarified. The goal of this study is to explore our understanding of the mechanism by which HCC may develop and progress through identification of the role of epigenetically regulated miRNAs influences in the liver carcinogenesis. The current study included 60 patients who were well diagnosed as HCC patients. 60 patients who suffer from liver cirrhosis were also enrolled in the current study and 30 healthy control subjects who serve as control group. All patients will be subjected to: full clinical assessment, abdominal ultrasound, Blood sample will be withdrawn from every patients for both biochemical and serum detection of microRNAs (191-203 -335) by real time PCR. We found that all studied microRNAs were down regulated among HCC patients when compared to cirrhotic patients and controls (p value: 0.005, 0.005 and 0.001 for microRNAs 191, 203 and 335 respectively). Moreover, these microRNAs can discriminate cases of HCC from risky cirrhotic patients. We can conclude that downregulated microRNAs among HCC cases proposed a pattern to explain the role of DNA methylation on miRNA and gene expression in HCC.

Serum apelin levels and metabolic risk markers in obese women.

BACKGROUND: Adipose tissue hormones, Adipokines, play an important role in obesity-associated complications. Apelin has recently been added to the family of adipokines. The aim of this study was to evaluate the relationship between serum apelin levels and metabolic abnormal parameters in Egyptian obese women. MATERIALS AND METHODS: The study included 400 unrelated women; they were 200 obese women and 200 non- obese matched healthy women. All participants underwent clinical, anthropometric and biochemical examinations. Insulin resistance (IR) was determined by the homeostasis model assessment of insulin resistance (HOMA-IR). Serum apelin levels and obesity biomarkers were measured using enzyme-linked immunoassay (ELISA) kits. Fat mass was measured by Tanita Body Composition Analyzer. RESULTS: Obese women showed significant higher levels of serum apelin, leptin, triglycerides, LDL-C, total cholesterol, fasting insulin HOMA-IR and blood pressure levels than controls. Significant positive correlations between apelin and leptin levels with abnormal metabolic markers were noted in obese women. CONCLUSION: The present study suggests the significant role that might be mediated by apelin for developing abnormal metabolic parameters among Egyptian obese women.

Diagnostic Accuracy of Acoustic Radiation Force Impulse (ARFI) in Diagnosis of Liver Fibrosis among Egyptian Patients with Chronic HCV Infection.

BACKGROUND: Acoustic radiation force impulse (ARFI) evaluates liver stiffness non-invasively and was invented recently. This technique can easily and accurately assess the degree of liver fibrosis in clinical practice. AIM: The aim of this study was to detect the diagnostic performance of ARFI elastography in the staging of fibrosis in some Egyptian patients with chronic HCV infection. PATIENTS AND METHODS: One hundred ninety patients with chronic HCV infection; 142 men and 48 women were enrolled in the study. They underwent liver biopsy examination for liver fibrosis detection. All demographic; clinical and biochemical data were recoded. ARFI examination was done for all subjects to detect liver stiffness measurement in relation to liver fibrosis detected by pathological examination of liver biopsies. RESULTS: Medians of liver stiffness measurement by shear wave velocity showed a significant increase as a grade of liver fibrosis increases (p ≤ 0.0001, highly significant). Liver stiffness was directly correlated to age, AST; ALT; INR and liver steatosis (p values were: 0.009; 0.0001; 0.013; 0.006 and 0.04 respectively, significant). On the other hand, liver stiffness was inversely correlated to albumin; prothrombin concentration and platelets (p values were: 0.0001; 0.001, and 0.0001, respectively, significant). We found that shear wave velocity can predict F1; F2; F3 and F4 at cut-off values: 1.22; 1.32; 1.44 and 1.8 respectively. CONCLUSION: ARFI is a diagnostic noninvasive promising technique for liver fibrosis diagnosis among Egyptian patients with chronic HCV infection.

Relationship Between Serum microRNA155 and Telomerase Expression in Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Activation of telomerase reverse transcriptase (hTERT) has a role in liver carcinogenesis where telomerase is normally suppressed in most human somatic tissues after birth. In the current study we aimed to detect the significance of hTERT mRNA in early detection of hepatocellular carcinoma (HCC) and to determine the relationship between serum microRNA155 and telomerase expression. METHODS: Serum and liver tissue samples were collected from 40 patients (17 samples from patients with liver cirrhosis and 23 samples from patients with HCC) and 12 blood samples from healthy subjects were collected. Serum miRNA155 levels and blood and tissue hTERT mRNA were detected by real-time quantitative reverse-transcriptase PCR (RT-qPCR) among liver cirrhosis and HCC patients. Moreover, miR-155, blood and tissue hTERT levels were analyzed in relation to clinical and pathological features. RESULTS: Calculated expression of miRNA155 revealed that relative quantity (RQ) miR 155 was overexpressed in sera of HCC patients when compared to patients with liver cirrhosis and controls (p <0.0001). The median values of serum telomerase were significantly increased among HCC patients than in patients with liver cirrhosis and controls (p = 0.04). Moreover, tissue expression of telomerase was significantly higher in malignant tissue more than adjacent nonmalignant tissue among HCC patients (p = 0.02). It was also found that tissue expression of telomerase was significantly decreased in tissue of liver cirrhosis patients (p = 0.03). Interestingly, we found that blood telomerase was directly correlated with serum miRNA155 (p = 0.003). CONCLUSIONS: Both mir 155 and telomerase may have a role in development of HCC and mir 155 could regulate telomerase expression during liver carcinogenesis.

Relation between microRNAs and Apoptosis in Hepatocellular Carcinoma.

AIM: To determine the relation between serum microRNAs and apoptotic markers as regards development of HCC to understand the underlying mechanism of HCV related hepatocarcinogenesis. PATIENTS AND METHODS: A total of 65 serum samples (25 samples from controls, 20 samples from hepatitis and 20 samples from HCC patients) were collected for miRNAs (mir 21, mir 199-a, and mir 155) detection. Human Programmed cell death protein-4 (PDCD-4) and Human Cytochrome-C (CYT-C) were determined. RESULTS: miRNAs 21 and 155 were over expressed in sera of patients with HCC compared to patients with chronic hepatitis (p < 0.0001). While serum means values of miR 199a was significantly decreased among HCC group patients when compared to patients with chronic hepatitis (p < 0.0001). The serum levels of PCDC4 and CYTC were increased in patients with HCC when compared to chronic hepatitis patients. They were also increased in patients with chronic hepatitis when compared to controls (p < 0.05, significant). There was direct correlations between apoptotic markers and oncomirs miRNAs 21 and 155 while apoptotic markers were inversely correlated with miRNA 199-a. CONCLUSION: Both microRNAs and apoptotic markers have roles in HCC pathogenesis. It seems that oncogenic microRNAs induce liver carcinogenesis in HCV patients irrespective of suppression of apoptosis.

The potential role of miRNAs 21 and 199-a in early diagnosis of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is regarded as one of the most common malignancies and among the leading causes of cancer death among the whole world. The most urgent needs are to find sensitive markers for early diagnosis for HCC. MicroRNAs (miRNAs) are reported as a group of small non-coding RNAs that can function as endogenous RNA interference to regulate expression of the targeted genes. This study was conducted to detect the serum and tissue expression of miR 21 and miR 199-a to be applied as early detectors for HCC. METHODS: A total of 40 serum and tissue samples (17 samples from chronic hepatitis and 23 samples from HCC patients) were collected. The levels of the two mature miRNAs (miR-21 and miR-199-a) were detected by real time quantitative reverse-transcriptase PCR (RT-qPCR) in sera and tissues of chronic hepatitis and HCC patients. Besides, miR-21 and miR-199-a levels in relation to clinical and pathological factors were explored. RESULTS: We found that the expression of serum miR-21 was distinctly increased in HCC compared with chronic hepatitis (P<0.001). miR 199-a was distinctly decreased in HCC compared with chronic hepatitis (P<0.001). In addition, median of miR 21 was increased in malignant when compared to adjacent non-malignant tissues without significant differences (P=0.191) while miR 199-a was significantly decreased in malignant when compared to adjacent nonmalignant tissues (P<0.001). ROC analysis showed that miR-21 and miR-199-a might be potential biomarkers for HCC. CONCLUSIONS: In conclusion, the expression of miR-21 was significantly up-regulated and miR-199-a was significantly down regulated in serum of patients with HCC. Due to their reasonable sensitivity and specificity for disease progression, miR-21 and miR-199-a could be used as potential circulating biomarkers for HCC.

The possible role of Cytochrome c and programmed cell death protein 4 (PDCD4) on pathogenesis of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is regarded as one of the most common malignancies and among the leading causes of cancer death in the whole world. Apoptosis is a fundamental process controlling cell death and plays a critical role in normal development of multicellular organisms. When abnormalities occur in apoptosis, a variety of diseases are caused, including cancer. The aim of the current study was to determine the serum expression of Cytochrome c and PDCD4 among patients with hepatocellular carcinoma and chronic hepatitis. PATIENTS AND METHODS: A total of 40 serum and tissue samples (17 samples from chronic hepatitis and 23 samples from HCC patients) were collected. Apoptotic markers in serum were carried out using the quantitative sandwich enzyme immunoassay technique. RESULTS: We found that serum levels of PCDC4 and Cytochrome c were increased in patients with HCC when compared to chronic hepatitis patients. They were also increased in patients with chronic hepatitis when compared to controls (p < 0.05, significant). Analyzing the impact of HCC characters on serum values of PDCD4 and Cytochrome c revealed that the mean values of both PDCD4 and Cytochrome c are significantly higher in cases with single lesion of HCC (p < 0.05, significant). Right lobe location of HCC lesions has the highest mean values of PDCD4 (p < 0.05, significant). As regards grade of differentiation, grade П has higher mean values of Cytochrome c (p < 0.05, significant). CONCLUSION: Serum levels of Cytochrome c and PDCD4 are increased in patients with cirrhosis and hepatocellular carcinoma and could be used as diagnostic aid for HCC.

Possible role of angiotensin-converting enzyme polymorphism on progression of hepatic fibrosis in chronic hepatitis C virus infection.

Many functional polymorphisms in the rennin-angiotensin system (RAS) have been described; these polymorphisms have been postulated to contribute to fibrosis in several diseases. Our aim was to study the frequency of ACE I/D polymorphism in chronic hepatitis C virus (HCV) infection and its association with liver fibrosis and response to treatment. This study included 90 patients with chronic hepatitis C. All patients received antiviral therapy in the form of pegylated interferon and ribavirin. Patients were grouped according to the stage of liver fibrosis by biopsy into: group 1 (fibrosis: 0 or 1); group 2 (fibrosis: 2 or 3) and group 3 (fibrosis: 4 or 5). The study included also 170 healthy subjects, as a control group. Polymerase chain reaction was carried out to detect the different ACE genotypes. The D/D genotype was significantly more prevalent among HCV patients compared to controls (65.6% vs 48.2%, P=0.006). Degree of necroinflammation was significantly higher among patients with I/I genotype when compared to patients with D/D genotype (P=0.04). No significant difference in the distribution of the ACE I/D genotypes between the fibrosis groups and between responders and non responders to interferon therapy. The D/D genotype may increase the susceptibility to infection with hepatitis C.

The potential role of insulin-like growth factor 1, insulin-like growth factor binding protein 3 and bone mineral density in patients with chronic hepatitis C virus in Cairo, Egypt.

Insulin-like growth factor 1 (IGF-1) levels have been found to correlate with measurements of bone mineral density (BMD) in liver diseases. This study investigated the relationship between IGF-1, insulin-like growth factor binding protein 3 (IGFBP-3) and BMD in patients with chronic hepatitis C virus. This study was conducted for 30 patients with chronic hepatitis C virus infection (16 patients without and 14 patients with cirrhosis) and 11 healthy controls. Serum levels of IGF-1 and IGFBP-3 and BMD of the proximal femur and lumbar spine were measured in all subjects. Osteoporosis of the proximal femur and lumbar spine was found in 42.9% and 21.4%, respectively, of the patients with cirrhosis. Patients with liver cirrhosis and osteoporosis of the proximal femur and lumbar spine had lower IGF-1 (P<0.001, P=0.04, P=0.04 respectively). BMD of the proximal femur was lower in cirrhotic patients compared with controls (P<0.01). Patients with liver cirrhosis had lower IGFBP-3 than patients without cirrhosis and controls (P<0.001). Patients with osteoporosis of the proximal femur had lower IGFBP-3 than those without osteoporosis (P<0.01). IGF-1 and IGFBP-3 levels were lower in patients with liver cirrhosis. IGF-1 and IGFBP-3 may play a role in hepatic osteoporosis.