Circulating microRNA expression and nonalcoholic fatty liver disease in adolescents with severe obesity.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases in children and adolescents. NAFLD ranges in severity from isolated hepatic steatosis to nonalcoholic steatohepatitis (NASH), wherein hepatocellular inflammation and/or fibrosis coexist with steatosis. Circulating microRNA (miRNA) levels have been suggested to be altered in NAFLD, but the extent to which miRNA are related to NAFLD features remains unknown. This analysis tested the hypothesis that plasma miRNAs are significantly associated with histological features of NAFLD in adolescents. AIM: To investigate the relationship between plasma miRNA expression and NAFLD features among adolescents with NAFLD. METHODS: This study included 81 adolescents diagnosed with NAFLD and 54 adolescents without NAFLD from the Teen-Longitudinal Assessment of Bariatric Surgery study. Intra-operative core liver biopsies were collected from participants and used to characterize histological features of NAFLD. Plasma samples were collected during surgery for miRNA profiling. A total of 843 plasma miRNAs were profiled using the HTG EdgeSeq platform. We examined associations of plasma miRNAs and NAFLD features using logistic regression after adjusting for age, sex, race, and other key covariates. Ingenuity Pathways Analysis was used to identify biological functions of miRNAs that were associated with multiple histological features of NAFLD. RESULTS: We identified 16 upregulated plasma miRNAs, including miR-193a-5p and miR-193b-5p, and 22 downregulated plasma miRNAs, including miR-1282 and miR-6734-5p, in adolescents with NAFLD. Moreover, 52, 16, 15, and 9 plasma miRNAs were associated with NASH, fibrosis, ballooning degeneration, and lobular inflammation, respectively. Collectively, 16 miRNAs were associated with two or more histological features of NAFLD. Among those miRNAs, miR-411-5p was downregulated in NASH, ballooning, and fibrosis, while miR-122-5p, miR-1343-5p, miR-193a-5p, miR-193b-5p, and miR-7845-5p were consistently and positively associated with all histological features of NAFLD. Pathway analysis revealed that most common pathways of miRNAs associated with multiple NAFLD features have been associated with tumor progression, while we also identified linkages between miR-122-5p and hepatitis C virus and between miR-199b-5p and chronic hepatitis B. CONCLUSION: Plasma miRNAs were associated with NAFLD features in adolescent with severe obesity. Larger studies with more heterogeneous NAFLD phenotypes are needed to evaluate miRNAs as potential biomarkers of NAFLD.

[Cloning, Expression and Immungenicity Analysis of Antigen Eg-01883 Screened from Protoscoleces of Echinococcus granulosus].

Objective: To screen for the Echinococcus granulosus 01883（Eg-01883） specifically expressed at the protoscolex period, clone and express this molecule as well as analyse its immunogenicity. Methods: Eg-01883, which is highly expressed at the protoscolex period but not in oncosphere， was screened by analysing the published mRNA sequences of E. granuolosus. Total RNA of E. granuolosus was extracted, Eg-01883 was cloned by RT-PCR, and the recombinant plasmid pET28a-Eg-01883 was constructed. Expression of the recombinant protein rEg-01883 was induced by isopropyl-ß-D-thiogalactoside （IPTG）. ICR mice were randomized into 3 groups （n=12 in each group）. Mice in the immunization group received subcutaneous injections of 10 µg rEg-01883 in 100 µl PBS emulsified in Freund's adjuvant at multiple sites, followed by immune enhancement after 2 weeks. Mice in the adjuvant group were injected with PBS and adjuvant. Mice in the control group received no treatment. Blood was obtained through caudal vein before immunization, and at 1, 2, and 4 weeks after the first immunization, and through the eyeball at 6 weeks after immunization. Serum levels of IgG, IFN-γ and IL-4 were determined by ELISA. The immunogenicity of rEg-01883 was identified by Western blotting. Results: Eg-01883 was screened, cloned, expressed and purified to obtain the recombinant protein rEg-01883, which mainly existed as the inclusion body. ELISA results showed that immunization with rEg-01883 induced production of specific IgG antibody. The serum IgG level in the immunization group increased from 1 week after the first immunization, peaked at 6 weeks（2.344±0.153）, which was significantly higher than those of the adjuvant group（0.206 1±0.006） and the control group （0.241±0.01） （P<0.01）. At 6 weeks after the first immunization, the serum levels of IFN-γ （43.23 pg/ml） and IL-4（24.88 pg/ml） in the immunization group were significantly higher than those in the adjuvant group（21.77 pg/ml, 13.27 pg/ml） and the control group（17.40 pg/ml, 12.25 pg/ml）（P<0.05）. Western blot showed that the recombinant protein rEg-01883 could be recognized by His-Tag antibodies, serum of immunized mice, and serum of mice with secondary infection. Conclusion: The recombinant protein rEg-01883 shows good immunogenicity in ICR mice.