A total of 36 patients with Hepatitis B and liver fibrosis and 25 patients with Hepatitis B and liver cirrhosis admitted to the Linyi People's Hospital (Linyi, China) between June 2012 and February 2016 were included in the present study. Among the 36 patients with liver fibrosis, 20 were male and 16 were female, and they had a median age of 41 years (age range, 20–61 years). Among the 25 patients with liver cirrhosis, 15 were male and 10 were female, and they had a median age of 43 years (age range, 18–65 years). All patients were diagnosed with liver fibrosis or cirrhosis according to the guidelines for the prevention and treatment of chronic Hepatitis B (23). The levels of alanine aminotransferase and aspartate aminotransferase were determined in patients with liver fibrosis using an automatic biochemical analyzer (Beckman Coulter, Inc., Brea, CA, USA). The normal concentrations in the blood for alanine aminotransferase and aspartate aminotransferase levels were 0–40 and 5–40 U/l, respectively. The levels of alanine aminotransferase and aspartate aminotransferase in patients with liver fibrosis were 168.6±39.6 and 146.2±28.5 U/l, respectively. The levels of alanine aminotransferase and aspartate aminotransferase in patients with liver cirrhosis were 182.8±51.9 and 161.2±39.3 U/l, respectively. Patients with immune-related diseases or immune diseases, including diabetes and cancer, were excluded from the current study. Liver biopsies (1.0–1.5 cm in length) and peripheral blood samples (20 ml) were collected from each patient. Blood serum was isolated from peripheral blood following centrifugation at 400 × g for 10 min at 4°C. To obtain peripheral blood mononuclear cells (PMBCs), a mixture of heparin-anticoagulant venous blood and equal amount of serum-free Iscove's modified Dulbecco's medium (1:1 v/v) was added gently to the lymphocyte separation medium before centrifugation at 400 × g for 30 min at 4°C. After centrifugation, the middle layer was aspirated and mixed with 5 volumes of Hanks solution before centrifugation at 300 × g for 10 min at 4°C, washed twice and cell density was adjusted to 1×10⁶ cells/ml. Cells were seeded into six-well plates at a density of 3×10⁶ cells/well and incubated at 37°C in a 5% CO₂-humidified incubator for 1–2 h. The cells that attached after 1–2 h incubation were PBMCs. All procedures were approved by the Ethics Committee of Linyi People's Hospital and written informed consent was obtained from all patients or their families.

Prior to total RNA extraction, tissue samples (100 mg) were ground into powder using liquid nitrogen. Total RNA was extracted using 1 ml TRIzol^® (10606ES60; Shanghai Yeasen Biotechnology, Co., Ltd., Shanghai, China). Following lysis, miRNA was isolated using the miRcute miRNA isolation kit (Tiangen Biotech Co., Ltd., Beijing, China). The purity of RNA was determined at A260/A280 using an ultraviolet spectrophotometer (NanoDrop ND1000; Thermo Fisher Scientific, Inc., Wilmington, DE, USA). The TIANScript II cDNA First Chain Synthesis kit (KR107; Tiangen, Biotech, Co., Ltd., Beijing, China) was used to reverse transcribe 1 µg RNA into cDNA, which was stored at −20°C. The primer sequences used were as follows: IL-6 forward, 5′-GGCACTGGCAGAAAACAACC-3′ and reverse, 5′-GCAAGTCTCCTCATTGAATCC-3′; GAPDH, forward, 5′-GGGAAACTGCGGCGTGAT-3′ and reverse, 5′-AAAGGTGGAGGAGTGGGT-3′. The PCR reaction mixture (20 µl) contained 10 µl SuperReal PreMix SYBR-Green (Tiangen Biotech Co., Ltd.), 0.5 µl upstream primer, 0.5 µl downstream primer, 2 µl cDNA and 7 µl ddH₂O. The following thermocyclic conditions were used for the qPCR: Initial denaturation at 95°C for 30 sec; 45 cycles of 95°C for 5 sec and 57°C for 30 sec. The qPCR was performed on an iQ5 Real-Time PCR Detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The 2^−ΔΔCq method was used to calculate the relative expression of IL-6 mRNA against the reference gene GAPDH (24). For miR-146a, the primer sequences were as follows: miR-146a forward, 5′-CGGCGGTGAGAACTGAATTCCA-3′ and reverse, 5′-GTGCAGGGTCCGAGGT-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. The following thermocyclic conditions were used for the qPCR: Initial denaturation at 95°C for 5 min; 40 cycles of denaturation at 95°C for 10 sec, 60°C for 20 sec and 70°C for 10 sec. The qPCR was performed as above and the 2^−ΔΔCq method was used to calculate the relative expression of miR-146a against the reference gene U6.

Total protein was extracted from tissues and PBMCs using radioimmunoprecipitation assay lysis buffer (600 µl; 50 mM Tris-base, 1 mM EDTA, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% Triton X-100, 1% sodium deoxycholate; Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's protocol. Following lysis for 50 min on ice, the mixture was centrifuged at 13,400 × g and 4°C for 5 min. The supernatant was used to determine protein concentration using a bicinchoninic acid protein concentration determination kit [RTP7102; Real-Times (Beijing) Biotechnology Co., Ltd., Beijing, China]. Protein samples (20 µg) were then mixed with sodium dodecyl sulfate loading buffer prior to denaturation in a boiling water bath for 5 min. Afterwards, 50 µg protein/lane was separated via SDS-PAGE on a 10% gel. Resolved proteins were then transferred to polyvinylidene difluoride membranes on ice (100 V, 2 h) and blocked with 5% skimmed milk at room temperature for 1 h. Subsequently, membranes were incubated with rabbit anti-human IL-6 polyclonal primary antibody (1:1,000; ab6672) and rabbit anti-human β-actin primary antibody (1:5,000; ab129348; both Abcam, Cambridge, UK) at 4°C overnight. Following three extensive washes with phosphate-buffered saline with Tween 20 for 15 min each time, membranes were incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:3,000; ab6721; Abcam) for 1 h at room temperature prior to three washes with phosphate-buffered saline with Tween-20 for 15 min each time. Subsequently, the membrane was developed using an enhanced chemiluminescence detection kit (ab65623; Abcam) for imaging. Image lab v3.0 software (Bio-Rad Laboratories, Inc.) was used to acquire and analyze imaging signals. The relative content of IL-6 protein was expressed as the IL-6/β-actin ratio.

Serum samples were tested using an IL-6 ELISA kit (ab178013; Abcam). In microplates, standards (50 µl), samples (10 µl sample liquid and 40 µl diluent) and blank were added to predefined wells. In the wells for standards and samples, horseradish peroxidase-labeled conjugates (100 µl) were added prior to sealing the plates for incubation at 37°C for 1 h. Following five washes of the plates, substrates A (50 µl) and B (50 µl) were added to each well. Following incubation at 37°C for 15 min, stop solution (50 µl) was added to each well, and the absorbance of each well was measured at 450 nm within 15 min.

Bioinformatic predictions are a useful tool to use to evaluate miR function. To determine the regulation of IL-6 in liver fibrosis and cirrhosis, miRanda (www.microrna.org/rnicrorna/home.do), TargetScan (www.targetscan.org), PiTa (genie.weizmann.ac.il/pubs/mir07/mir07_data.html), RNAhybrid (bibiserv.techfak.uni-bielefeld.de/rnahybrid) and PICTA (pictar.mdc-berlin.de) were used to predict the miR molecules that regulate IL-6 and indicated that miR-146a may regulate IL-6 (Fig. 1). According to the bioinformatic results, wild-type (WT) and mutant seed regions of miR-146a in the 3′-untranslated region (UTR) of IL-6 were chemically synthesized in vitro, SpeI and HindIII restriction sites were added, and the WT and mutant miR-146a were cloned into pMIR-REPORT luciferase reporter plasmids (Ambion; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Plasmids (0.8 µg) with WT or mutant 3′-UTR DNA sequences were co-transfected with 100 nM agomiR-146a (Sangon Biotech, Shanghai, China) into 293T cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Following 24 h cultivation, cells were lysed using a dual luciferase reporter assay kit (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol and fluorescence intensity was measured using a GloMax 20/20 luminometer (Promega Corporation). Renilla fluorescence activity was used as internal reference to measure the fluorescence values of each group of cells.

The results were analyzed using SPSS 18.0 statistical software (SPSS, Inc., Chicago, IL, USA). The data are presented as the mean ± standard deviation and were tested for normality. Multigroup measurement data were analyzed using one-way analysis of variance. In the case of homogeneity of variance, Least Significant Difference and Student-Newman-Keuls methods were used; in the case of heterogeneity of variance, Tamhane's T2 or Dunnett's T3 method were used. P<0.05 indicated a statistically significant difference.

To measure the expression of IL-6 mRNA in the different samples, RT-qPCR was performed. The data indicated that levels of IL-6 mRNA in liver tissues, PBMCs and serum from patients with liver cirrhosis were significantly increased compared with those from patients with liver fibrosis (P<0.05; Fig. 2). These results suggest that the expression of IL-6 mRNA in patients with liver cirrhosis is higher than in patients with liver fibrosis.

To determine the expression of IL-6 protein in liver tissues and PBMCs, western blotting was performed. Compared with patients with liver fibrosis, the expression of IL-6 in liver tissues and PBMCs from patients with liver cirrhosis was significantly enhanced (P<0.05, P<0.01, respectively; Fig. 3). This was consistent the results from RT-qPCR. These results indicate that IL-6 exerts its regulatory effect on liver cirrhosis at the protein level.

To examine the content of IL-6 protein in the serum, ELISA was employed. The data demonstrated that IL-6 levels in the serum of patients with liver cirrhosis were significantly higher that in patients with liver fibrosis (P<0.05; Fig. 4). This suggests that increased IL-6 protein levels in serum may be released from PBMCs.

To measure miR-146a levels in various samples, RT-qPCR was performed. Compared with patients with liver fibrosis, miR-146a levels in the liver tissues, PBMCs and serum from patients with liver cirrhosis were significantly downregulated (P<0.05, P<0.05 and P<0.01, respectively; Fig. 5). These results indicate that altered IL-6 expression may be induced by alterations in miR-146a levels.

To identify the interaction between miR-146a and IL-6, a dual luciferase reporter assay was performed. The data demonstrated that the fluorescence value of the WT group was significantly lower than that of the NC (P<0.01), whereas that of the mutant group did not differ significantly from the control (P>0.05; Fig. 6). The results suggest that miR-146a regulates the expression of IL-6 by binding to its 3′-UTR.