313 CHB patients, 153 patients with other liver diseases, and 33 healthy controls were recruited from among out- or in-patients in the Department of Infectious Diseases and Health Management Center from December 8, 2017 to September 13, 2018, and were enrolled in this study. CHB and other liver diseases were diagnosed according to AASLD 2018 hepatitis B guidance and other pertinent diagnostic criteria.
Collection of serum samples and clinical biochemical tests
Serum samples were allowed to clot for two hours at room temperature (RT), then underwent centrifugation at 1000×g for 20 minutes, and supernatants were stored at -80℃ for later analysis. Clinical serological biochemical tests were carried out in the clinical laboratory of the Department of Infectious Diseases, Southwest Hospital.
Dynamic observation of SIX1 and EYA1 serum levels in selected CHB patients before and after comprehensive treatment
Serum samples collected from CHB patients whose alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were over 100 IU/L before antivirus regimens or non-specific therapy and below 100 IU/L after treatment respectively and were tested for SIX1 and EYA1 levels by ELISA. As such, these samples were different from previous samples even from the same patient.
Assessment of SIX1 and EYA1 serological levels by ELISA
Both SIX1 and EYA1 ELISA kits were purchased from Shanghai JiangLai Industrial Limited by Share Ltd., and were used according to provided instructions. Briefly, 50μl of standard or sample was added to appropriate wells, and then100μl of enzyme conjugate was added to wells for 60 minutes at37°C. Plates were then washed 4 times, and 50μl each of substrate A and B were added to each well for 15 minutes at 37°C protected from light. Next, 50μl stop solution was added to each well, and the optical density(O.D) of each well was measured at450 nm using a microplate reader (Multiskan Spectrum 51118650, Thermo Scientific, Waltham, MA, USA) within 15minutes.
Liver biopsy samples were obtained from a CHB inpatient. Human healthy liver tissues were provided by Xi’an Alenabio. Inc and were used as a positive control. B6 mouse liver was used as negative control.
Immunofluorescent staining for SIX1, EYA1, human serum albumin (HSA), and glial fibrillary acidic protein (GFAP) in frozen liver sections
Frozen sections were fixed with 100% ethanol for 15 minutes and permeabilized using 0.05% Tween20 twice, 2 minutes per treatment. Blocking was performed using 3% bovine serum albumin for 30 minutes at RT. After washing with PBS, sections were incubated with rabbit anti-human SIX1 primary antibody (1:100 dilution) (HPA001893, Sigma, St. Louis, MO USA), rabbit anti-human EYA1 primary antibody (1:100 dilution) (ab85009, abcam, Cambridge, MA, USA) and mouse anti-human HSA primary antibody (1:1000 dilution) (MAB1455, R&D Systems, Minneapolis MN, USA) or mouse anti-human GFAP primary antibody (1:100 dilution) (MA5-12023,ThermoFisher, Waltham, MA, USA) at 4°C overnight. Sections were then incubated with Alexa Fluor®488 donkey anti-mouse IgG and Alexa Fluor®568 donkey anti-rabbit IgG (1:1000 dilution) (A21202, A10042, ThermoFisher, Waltham, MA, USA) for one hour at RT. After washing with PBS, sections were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). Finally, coverslips were mounted with anti-fade mounting medium, and immunofluorescent signals were visualized and recorded using an Olympus DP72 microscope and the cellSens Standard 1.5 software.
Protein-protein interaction (PPI) network analysis
A PPI network was constructed using the Search Tool for the Retrieval of Interacting Genes (https://string.embl.de/) database with a medium confidence of 0.40. Combined scores were calculated based on neighborhood, experiment, gene fusion, co-occurrence, text mining, co-expression, and database annotations.
Statistical analyses were carried out using the SPSS v17.0 statistical package (SPSS Inc., Chicago, IL, USA), with comparisons being made via student’s t-tests. P<0.05 was considered to be statistically significant.