Nanoconjugated long non-coding RNA MEG3 as a new therapeutic approach for Hepatocellular carcinoma.

Background: Hepatocellular carcinoma (HCC) is an aggressive human cancer with a poor prognosis. Long non-coding RNAs (lncRNA) have multiple functions: epigenomic regulation, gene transcription, protein-coding gene translation, and genome defense. The involvement of lncRNAs in therapy offers a vast step in cancer treatment. Objective: In the current study, a novel therapeutic regimen using polymer nanoparticle-mediated delivery of lncRNA was designed to control the progression of hepatocarcinogenesis. Methods: One hundred mice were divided into 5 groups. The first group served as a normal-control group and was injected with saline, whereas the pathological-control group (the second group) was injected with N-Nitrosodiethylamine (DEN) weekly for 16 weeks. Group 3, Group 4, and Group 5 were injected intrahepatically with polymer nanoparticles (NPs) alone, lncRNA MEG3 alone, and conjugated NPs, respectively, once/week for four weeks starting on the 12th week after DEN injection. After 16 weeks, animals were euthanized, and liver specimens and blood samples were collected for pathological, molecular, and biochemical assessment. Results: Compared to the pathological-control group, nanoconjugates lncRNA MEG3 demonstrated a significant improvement in histopathology and tumour-associated biomarkers. Furthermore, the expression of the SENP1 and PCNA was downregulated. Conclusion: MEG3 conjugated nanoparticles can be considered a novel therapeutic regimen for HCC.

Development of liver microtissues with functional biliary ductular network.

Liver tissue engineering aims to create transplantable liver grafts that can serve as substitutes for donor's livers. One major challenge in creating a fully functional liver tissue has been to recreate the biliary drainage in an engineered liver construct through integration of bile canaliculi (BC) with the biliary ductular network that would enable the clearance of bile from the hepatocytes to the host duodenum. In this study, we show the formation of such a hepatic microtissue by coculturing rat primary hepatocytes with cholangiocytes and stromal cells. Our results indicate that within the spheroids, hepatocytes maintained viability and function for up to 7 days. Viable hepatocytes became polarized by forming BC with the presence of tight junctions. Morphologically, hepatocytes formed the core of the spheroids, while cholangiocytes resided at the periphery forming a monolayer microcysts and tubular structures extending outward. The spheroids were subsequently cultured in clusters to create a higher order ductular network resembling hepatic lobule. The cholangiocytes formed functional biliary ductular channels in between hepatic spheroids that were able to collect, transport, and secrete bile. Our results constitute the first step to recreate hepatic building blocks with biliary drainage for repopulating the whole liver scaffolds to be used as transplantable liver grafts.

Impact of E-cadherin and its transcription regulators on assessing epithelial-mesenchymal transition in chronic hepatitis C virus infection.

BACKGROUND: The mechanisms of chronic hepatitis C virus (HCV)-induced liver fibrosis and hepatocarcinogenesis are still poorly recognized. Therefore, this study aimed to determine the effect of chronic HCV infection on the expression of the major regulators of epithelial-mesenchymal transition (EMT) including E-cadherin, Snail, Slug, and Twist2, in the Egyptian population. This will help to design more efficient strategies to treat HCV-associated cirrhosis and carcinoma. METHODS: Fifty-nine liver biopsies from patients, that were serologically proven to be HCV positive, were included in the current study. Histopathological examination was done. Grading of hepatitis activity (A) and staging of fibrosis (F) were assessed using the METAVIR Scoring System. Additionally, an immunohistochemical examination of E-cadherin, Snail, Slug, and Twist2 expression was performed. RESULTS: E-cadherin showed a significant progressive decline of its expression with increased fibrosis staging and development of hepatocellular carcinoma (HCC). In contrast, Snail and Slug expression was positively associated with the stage of fibrosis and HCC. Meanwhile, Twist2 expression was not affected by the degree of hepatitis activity, the stage of fibrosis, or by the development of HCC. CONCLUSIONS: E-cadherin and its transcriptional regulators; Snail and Slug may serve as indicators for assessing the stage of fibrosis and the progression of HCC associated with HCV infection but not for assessing the degree of hepatitis activity. Therefore, the Snail family could be a promising target for designing effective preventive and therapeutic strategies for chronic HCV infection and its serious comorbidities.

MicroRNA-195 vector influence on the development of gradually induced hepatocellular carcinoma in murine model.

Background: Recent studies implicate the role of microRNAs in the pathogenesis of hepatocellular carcinoma (HCC). This study was designed to induce HCC, in an experimental model, with the prospect to study the molecular pathophysiologic changes accompanying the development of HCC and the effect of miRNA-195 vector on the process of hepatocarcinogenesis.Methodology: This study incorporated three groups of male albino mice; one control group and two other groups injected intraperitoneal with diethylnitrosamine (DEN) weekly for 12 weeks for the gradual induction of HCC. The third group was injected intra-hepatic with miR-195 vector 1 month after DEN injection. At the 8th and 12th weeks post-DEN treatment, the tumor-associated biomarkers alpha-fetoprotein (AFP), vascular endothelial growth factor (VEGF), and tumor necrosis factor-alpha (TNF-α) were assessed in the serum of all mice. Hepatic specimens were subjected to ultra-structural pathological examination as well as to caspase-3 and survivin genes expression analysis.Results: All the assessed serological and molecular parameters of HCC development, in the miRNA-195-treated group of mice, showed a significant increase, versus the DEN-treated group, whereas survivin was significantly down-regulated, in the miR-195-treated group (P < 0.001). Additionally, ultra-structural criteria of HCC were depicted, in the 12th week, in DEN-injected group, versus the 8th week, in the miRNA-195-treated group.Conclusions: Intra-hepatic injection of miRNA-195 vector induced apoptotic gene expression and suppressed anti-apoptotic gene but these favorable anti-cancer effects could not counteract the inflammatory, and subsequently, the oncogenic effect probably caused by vector administration. Therefore, further studies are required to investigate the effect of miRNA in combination with anti-inflammatory medications.

MicroRNA-122a as a non-invasive biomarker for HCV genotype 4-related hepatocellular carcinoma in Egyptian patients.

INTRODUCTION: Hepatitis C virus (HCV) infection persists in most infected individuals and can lead to the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) have a crucial role in various liver diseases, especially HCC. The expression profiles of circulating microRNAs have been studied aiming at the identification of novel non-invasive biomarkers. This study aims to develop a non-invasive diagnostic tool based on measuring the serum levels of different miRNAs in order to detect HCV-induced HCC at the early stages of the disease. MATERIAL AND METHODS: Five main miRNAs (miRNA-122a, miRNA-125a, miRNA-139, miRNA-145, and miRNA-199a) were selected according to the literature that demonstrated their unique expression pattern during HCC development. Serum samples were collected from 42 cases of chronic hepatitis C (CHC) without cirrhosis, 45 cases of CHC with cirrhosis (LC), 38 cases of HCC with HCV, and 40 healthy individuals serving as a control. The five miRNAs were measured using real-time reverse transcription PCR. The conventional HCC markers α-fetoprotein (AFP) and des-γ-carboxyprothrombin (DCP) were measured with commercial kits. RESULTS: Serum levels of miRNA-122a, miRNA-125a, miRNA-139, miRNA-145, and miRNA-199a were significantly lower (p < 0.01) in HCC than in CHC and LC groups. As a single marker, miRNA-122a had the highest sensitivity for HCC, followed by miRNA-199a, miRNA-145, miRNA-139, and miRNA-125a. CONCLUSIONS: These findings indicate that measurement of serum levels of miRNA-122a, miRNA-125a, miRNA-139, miRNA-145, and miRNA-199a can differentiate HCC from CHC and LC. Our results suggest that serum miR-122 might serve as a novel and potential noninvasive biomarker for HCV-induced HCC.

miRNA-221 and miRNA-222 are promising biomarkers for progression of liver fibrosis in HCV Egyptian patients.

BACKGROUND: Current methodologies used to determine the progression of hepatic fibrosis rely heavily on liver biopsy, a dangerous and invasive procedure, with semi-subjective analysis of the results of the biopsy. Thus, a new approach is immensely needed for monitoring the progression of liver fibrosis in Hepatitis C virus (HCV) patients. AIM OF WORK: The purpose of this study was to find highly specific and sensitive miRNA biomarkers that can be used to detect different stages of liver fibrosis. METHODOLOGY: The study consisted of 42 cases of chronic hepatitis C (CHC) with early-stage fibrosis, 45 cases of CHC with late-stage fibrosis, and 40 healthy subjects with no CHC or fibrosis as controls. Expression patterns of 5 miRNAs (miR-16, miR-146a, miR-214-5p, miR-221, and miR-222) were analyzed in each group using TaqMan real-time PCR. RESULTS: Serum levels of miRNA-16, miRNA-146a, miRNA-221, and miRNA-222 were all significantly up-regulated in early and late stages of liver fibrosis. miRNA-222 had the highest sensitivity and specificity values in early and late fibrosis. miRNA-221 had the second highest sensitivity and specificity with the late-stage fibrosis group. Furthermore, miRNA-221 showed significant positive correlations with both miRNA-16 and miRNA-146a in the early- and late-stage fibrosis groups, with the early stage having a stronger correlation. CONCLUSIONS: The results indicated that miRNA-16, miRNA-146a, miRNA-221, and miRNA-222 can be used to detect the presence of liver fibrosis. The high sensitivity and specificity of miRNA-222 and miRNA-221 in late-stage fibrosis indicate promising prognostic biomarkers for HCV-induced liver fibrosis.

Expression analysis of liver-specific circulating microRNAs in HCV-induced hepatocellular Carcinoma in Egyptian patients.

OBJECTIVES: Due to the absence of reliable and accurate biomarkers for the early detection of liver malignancy, circulating microRNAs have recently emerged as great candidates for prompt cancer identification. Therefore, the aim of this study was to investigate the potential of liver-specific circulating microRNAs as an accurate non-invasive diagnostic tool for early diagnosis of hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC). METHODOLOGY: A total of 165 patients were enrolled in this study and categorized into four main groups: 42 chronic hepatitis C (CHC) without cirrhosis, 45 CHC with cirrhosis (LC), 38 HCC with HCV patients, and 40 healthy controls. The expression profiles of seven miRNAs (miR-16, miR-34a, miR-125a, miR-139, miR-145, miR-199a, and miR-221) were analyzed using real-time PCR. RESULTS: Serum levels of miRNA-125a, miRNA-139, miRNA-145, and miRNA199a were significantly lower (p < 0.01) in HCC than in both CHC and LC groups. On the other hand, no significant difference was shown in the expression of miR-16, miR-34a, and miR-221 between the CHC, LC, and HCC groups. MiR-16, miR-34a, and miR-221 were significantly elevated in the HCC group compared to the control group. MiR-34a showed the highest specificity and sensitivity. CONCLUSIONS: The results indicated that the measurement of serum levels of miR-125a, miR-139, miR-145, and miR-199a can help to differentiate HCC from CHC and LC. Also, miR-16, miR-34a, and miR-221 serum levels would have a prognostic value. MiR-34a had the highest specificity and sensitivity, indicating that it might serve as a novel and potential non-invasive biomarker for HCV-induced HCC.

Performance of Disease-Specific Scoring Models in Intensive Care Patients with Severe Liver Diseases.

INTRODUCTION: Egypt has the highest prevalence of Hepatitis C Virus (HCV) in the world, estimated nationally at 14.7%. HCV treatment consumes 20% ($80 million) of Egypt's annual health budget. Outcomes of cirrhotic patients admitted to the ICU may, in fact, largely depend on differences in the state of the disease, criteria and indications for admission, resource utilization, and intensity of treatment. AIM: The aim of the present study was to evaluate the efficacy of liver specific scoring models in predicting the outcome of critically ill cirrhotic patients in the ICU as it may help in prioritization of high risk patients and preservation of ICU resources. MATERIALS AND METHODS: Over one year, a total of 777 patients with End Stage Liver Disease (ESLD) due to HCV infection were included in this retrospective non-randomized human study. All statistical analyses were performed by the statistical software SPSS version 22.0 (SPSS, Chicago, IL, USA). Child Turcotte Pugh (CTP) score, MELD score, MELD-Na, MESO, iMELD, Refit MELD and Refit MELD-Na were calculated on ICU admission. RESULTS: ICU admission was mainly due to Gastrointestinal (GI) bleeding and Hepatic Encephalopathy (HE). Overall mortality was 27%. Age and sex showed no statistical difference between survivors and non survivors. Significantly higher mean values were observed for all models among individuals who died compared to survivors. MELD-Na was the most specific compared to the other scores. MELD-Na was highly predictive of mortality at an optimized cut-off value of 20.4 (AURC=0.789±0.03-CI 95%=0.711-0.865) while original MELD was highly predictive of mortality at an optimized cut-off value of 17.4 (AURC=0.678±0.01-CI 95%=0.613-0.682) denoting the importance of adding serum sodium to the original MELD. INR, serum creatinine, bilirubin, white blood cells count and hyponatremia were significantly higher in non survivors compared to survivors, while hypoalbuminemia showed no statistical difference. The advent of Hepatorenal Syndrome (HRS) and Spontaneous Bacterial Peritonitis (SBP) carried worse prognosis. Hyponatremia and number of transfused blood bags were additional independent predictors of mortality. CONCLUSION: In cirrhosis of liver, due to HCV infection, patients who died during their ICU stay displayed significantly higher values on all prognostic scores at admission. The addition of sodium to MELD score greatly improves the predictive accuracy of mortality. MELD-Na showed the highest predictive value of all scores.

Bioactive chemical constituents of Curcuma longa L. rhizomes extract inhibit the growth of human hepatoma cell line (HepG2).

The present study was designed to identify the chemical constituents of the methanolic extract of Curcuma longa L. rhizomes and their inhibitory effect on a hepatoma cell line. The methanolic extract was subjected to GC-MS analysis to identify the volatile constituents and the other part of the same extract was subjected to liquid column chromatographic separation to isolate curcumin. The inhibition of cell growth in the hepatoma cell line and the cytopathological changes were studied. GC-MS analysis showed the presence of fifty compounds in the methanolic extract of C. longa. The major compounds were ar-turmerone (20.50 %), ß-sesquiphellandrene (5.20 %) and curcumenol (5.11 %). Curcumin was identified using IR, 1H and 13C NMR. The inhibition of cell growth by curcumin (IC50 = 41.69 ± 2.87 µg mL-1) was much more effective than that of methanolic extract (IC50 = 196.12 ± 5.25 µg mL-1). Degenerative and apoptotic changes were more evident in curcumin- treated hepatoma cells than in those treated with the methanol extract. Antitumor potential of the methanolic extract may be attributed to the presence of sesquiterpenes and phenolic constituents including curcumin (0.051 %, 511.39 µg g-1 dried methanol extract) in C. longa rhizomes.

Studies on the effect of pollution on Lake Manzala ecosystem in Port-Said, Damietta and Dakahlia Governorates Egypt.

This work studied how pollution impacts the ecosystem of Lake Manzala by determination of physicochemical parameters, studying biodiversity of aquatic plants and macroinvertebrates, and determining bioaccumulation of Pb, Cu, Cd & Zn in some major organisms, Biomphalaria alexandrina and Melanoides tuberculata snails and Oreochromis niloticus fish. The more near to Mediterranean Sea and to the industrial area, Port-Said and Damietta sites showed higher dissolved oxygen and conductivity than Dakahlia sites. Distribution percentage of Eichhornia crassipes is high among Port-Said and Dakahlia sites of 100 and 88%, respectively, while Lemna giba is the most abundant among Damietta sites of 60%. The maximum macroinvertebrate taxa richness was obtained at Gammalya, Dakahlia of 16 species while the maximum abundance was registered at Annanya, Damietta of 591 organisms. Gastropoda are the most distributed organisms in Lake Manzala followed by Hemiptera and Plecoptera then shrimps and scud. All the medically important snails, B. alexandrina, B. truncatus and L. natalensis were recorded in Dakahlia, but only B. alexandrina was in Damietta and Port-Said sites. The collected water samples from Damietta sites showed the highest significant Cu & Cd concentration while Port-Said samples showed the highest Pb concentration and Dakahlia showed the highest Zn concentration. The metals concentrations were higher in snail tissue and in fish liver, kidney and most of muscle samples as compared in surface water. The higher metal bioaccumulation was determined in snails collected from sites showed higher water metals concentrations. Fish muscle showed the least residues than liver and kidney for all the measured metals. Pb and Cd were more accumulated in kidneys, Cu was more accumulated in liver and Zn was accumulated in all examined fish parts in descending order as follows Kidney > liver > muscle.