Optimization of the replication of hepatitis E virus genotype 3 in vitro.

AIMS: Hepatitis E virus (HEV) is responsible for ∼20 million human infections worldwide every year. The genotypes HEV-3 and HEV-4 are zoonotic and are responsible for most of the autochthonous HEV cases in high-income countries. There are several cell culture systems that allow for propagation of different HEV genotypes in vitro. One of these systems uses human lung carcinoma cells (A549), and was further optimized for propagation of HEV-3 47832c strain. In this study, we investigated the effect of different media supplements as well as microRNA-122 (miR-122) on improving the replication of HEV-3 47832c in A549 cells. METHODS AND RESULTS: We observed that supplementation of maintenance media with 5% fetal bovine serum was sufficient for efficient replication of HEV-3, and verified the positive effect of media supplementation with Amphotericin B, MgCl2, and dimethyl sulfoxide on replication of HEV-3. We have also demonstrated that adding miR-122 mimics to the culture media does not have any significant effect on the replication of HEV-3 47832c. CONCLUSIONS: Herein, we detected over a 6-fold increase in HEV-3 replication in A549/D3 cells by adding all three supplements: Amphotericin B, MgCl2, and dimethyl sulfoxide to the culture media, while demonstrating that miR-122 might not play a key role in replication of HEV-3 47832c.

Evaluation of miR-122 to Predict High Dose Acetaminophen-Induced Liver Injury in Mice: The Combination Uses of 5-Fluorouracil.

Administration of high doses of acetaminophen (APAP) is known to cause drug-induced liver injury (DILI) in humans. Therefore, the detection or prediction of these side-effects at an early stage using appropriate biomarkers is the need of the hour. Micro RNA (miR)-122 is expected to be a novel biomarker for liver injury. However, more evidence is required in various alternate situations such as its use in combination as APAP is often used along with anticancer drugs. In the present study, we aimed to evaluate the functions of miR-122 as a biomarker for liver injury in comparison with alanine aminotransferase (ALT) in a mice model with the APAP-induced liver injury (AILI). Consequently, there was a dose-dependent increase in miR-122 after administration of APAP intraperitoneally. Similar observations were made for ALT activity. Additionally, the expression of miR-122 increased in a more rapid manner compared to ALT activity. However, there was a variation in the miR-122 expression. Further, we investigated the drug-drug interaction between APAP and 5-fluorouracil using miR-122 and ALT in mice. As a result, the degree of AILI was not changed by the use of 5-fluorouracil in combination with APAP in mice.

Significance of serum and hepatic microRNA-122 levels in patients with non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is believed to be a type of metabolic syndrome. MicroRNA-122 (miR-122) is the most abundant microRNA in the liver and is an important factor for the metabolism of glucose and lipids. In the present study, we examined the correlation between the hepatic and serum miR-122 expression levels and the clinicopathological factors of patients with NAFLD. METHODS: We extracted the total RNA, along with preserved miRNAs, from liver biopsy samples of 67 patients with NAFLD. In 52 of these 67 patients, the total RNA was extracted from serum. The miR-122 that was obtained by quantitative reverse transcription-polymerase chain reaction was quantified using TaqMan MicroRNA assays. RESULTS: A significant correlation was detected between serum and hepatic miR-122 expression (correlation coefficient, 0.461; P=0.005). Patients with mild steatosis (<33%) showed significantly lower levels of hepatic miR-122 compared with patients with severe steatosis (>33%) (hepatic miR-122: mild/severe=2.158±1.786/4.836±7.506, P=0.0473; serum miR-122: mild/severe=0.002±0.005/0.007±0.001, P=0.0491). Moreover, hepatic and serum miR-122 levels were significantly higher in patients with mild fibrosis than in those with severe fibrosis (hepatic miR-122: mild/severe=5.201±7.275/2.394±1.547, P=0.0087; serum miR-122: mild/severe=0.008±0.011/0.002±0.004, P=0.0191). CONCLUSIONS: We found that the hepatic and serum miR-122 levels were associated with hepatic steatosis and fibrosis. The serum miR-122 level can be a useful predictive marker of liver fibrosis in patients with NAFLD.

Pro-Neurotensin as a Potential Novel Diagnostic Biomarker for Detection of Nonalcoholic Fatty Liver Disease.

Background and Aims: Currently, liver biopsy is the gold standard method for diagnosis of non-alcoholic fatty liver severity. It is critical to develop non-invasive diagnostic method to diagnose nonalcoholic fatty liver rather than invasive techniques. Our case-control study was to address the value of circulating miRNA-122 and serum pro-neurotensin as a potential non-invasive biomarker for the diagnosis of non-alcoholic fatty acid diseases. Methods: Clinical assessment, laboratory investigations, and anthropometric measurements were reported for 157 patients with proven NAFLD. Apparently, healthy participants (n=100) were enrolled as a control group. Serum samples were tested for micro-RNAs-122 and pro-neurotensin. Results: Compared with the control subjects, both mi-RNA-122 and serum proneurotensin levels were increased in NAFLD (p<0.001) and at a cut-off ≥6.83, mi-RNA-122 had 51.0% sensitivity, 70.0% specificity to differentiate NAFLD from healthy controls, while serum proneurotensin had 80.0% sensitivity and 80.0% specificity at a cutoff ≥108. Conclusion: The circulating pro-neurotensin might be used as a novel biomarker for diagnosis of patients with NAFLD, wherefore the integration of a circulating mi-RNA-122 and serum pro-neurotensin could be beneficial to diagnose NAFLD cases. Large-scale studies are needed to investigate the possible role of mi-RNA-122 and pro-neurotensin in the development, progression, and prognosis of NAFLD and NASH.

Early diagnostic evaluation of miR-122 and miR-224 as biomarkers for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the common lethal types of tumor all over the world. The lethality of HCC accounts for many reasons. One of them, the lack of reliable diagnostic markers at the early stage, in this context, serum miRNAs became promising diagnostic biomarkers. Herein, we aimed to identify the predictive value of two miRNAs (miR-122 and miR-224) in plasma of patients with HCC preceded by chronic HCV infection. Taqman miRNA assays specific for hsa-miR-122 and hsa-miR-224 were used to assess the expression levels of the chosen miRNAs in plasma samples collected from three groups; 40 patients with HCC related to HCV, 40 with CHC patients and 20 healthy volunteers. This study revealed that the mean plasma values of miRNA-122 were significantly lower among HCC group when compared to CHC and control groups (P < 0.001). Whereas, miR-224 mean plasma values were significantly higher among HCC group when compared to both CHC group and control group. Moreover, it was found that miR-122 can predict development of HCC at cut-off value <0.67 (RQ) and (AUC = 0.98, P < 0.001). As regards miR-224, it can predict development of HCC at cut-off value >1.2 (RQ) and (AUC = 0.93, P < 0.001), while the accuracy of AFP to diagnose HCC was (AUC: 0.619; P = 0.06). In conclusion, the expression plasma of miR-122 and miR-224 could be used as noninvasive biomarkers for the early prediction of developing HCC at the early stage.