Construction of CMKLR1 gene interference plasmid vector
The specific interference sequence was designed according to the mRNA sequence of human CMKLR1 gene. The upstream and downstream sequences were introduced into the enzyme digestion site respectively. The oligo-DNA was synthesized and connected with the double enzyme digestion pGPSV plasmid vector. The connection accuracy was identified by sequencing.
Cell culture, transfection, and detection
Human monocyte U937 cells were cultured in RPMI Medium 1640 supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) at 37°C in a 5% CO2. CMKLR1 interfering plasmid and/or miRNA-4804-3p mimics were transfected into U937 cells using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) following the manufacturer’s protocol. Cell culture supernatants and cells were collected 48 h after addition of inhibitors or transfection. The cells were analyzed by immunofluorescence, quantitative PCR, or western blot. The levels of Interleukin(IL)-2, IL-10, Tumor necrosis factor(TNF)-α and Interferon(IFN)-γ in cell culture supernatant were detected by using a flow cytometry microsphere method.
Quantitative PCR detection of miRNA-4804-3p, CMKLR1, ERK, and NF-κB
Total RNA from cells was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA). The primers for miRNA-4804-3p, CMKLR1, ERK, NF-κB, U6, and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed and synthesized by Shanghai Bioengineering Co., Ltd. (Table 1). First-step cDNA synthesis was made using reverse transcription. The reaction conditions were 25 ℃ for 5 min, 42 ℃ for 60 min and 70 ℃ for 5min. Quantitative PCR (RT-PCR) was performed using 95°C for 10 min , followed by 95°C for 15 s and 60°C (collect fluorescence) for 60 s for 40 cycles. RT-PCR reactions were carried out for each sample and internal reference gene. GAPDH and U6 were used as internal standards.
Western blot
Cells were collected by adding 0.25% trypsin EDTA and centrifuged. The samples were mixed with 5 × SDS-PAGE loading buffer, and a total of 10 μL was heated at 100°C for 5 min. Insoluble precipitation were removed by centrifugation. The samples were separated by 8% SDS-PAGE. Proteins were transferred to PVDF membranes using a wet blotter. Membranes were blocked in 5% BSA, and polyclonal antibodies specific for CMKLR1, phosphorylated ERK (p-ERK), or NF-κB (Proteintech, Wuhan, China) were added and incubated overnight at 4°C. Membranes were washed and blocked with 5% BSA, followed by probing with secondary antibodies (Proteintech, Wuhan, China) at room temperature for 2 h. A Fr-1800 fluorescence biological image analysis system(Shanghai Furi Science & Technology Co., Ltd. China) was used for chemiluminescence detection. The gray value of each specific band was digitized and quantified by ImageJ analysis software (NIH, Bethesda, MD, USA) (https:// imagej.en.softonic.com).
Collection and detection of samples from patients with CHB
CHB outpatient and inpatient samples were collected. These patients were identified in accordance with the “Guidelines for the Prevention and Treatment of Chronic Hepatitis B” of China (2015 Edition) [15]. Exclusion criteria were as follows: (1) patients with hepatitis A virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, or HIV; (2) patients abusing alcohol, drugs, or having fatty liver disease; (3) patients with decompensated cirrhosis and liver tumors; (4) patients with important organ diseases (heart, brain, kidney, or diabetes). Written informed consent was obtained from all subjects. The experimental protocol was approved by the ethical commission of Taizhou Hospital of Zhejiang Province.
Collection and detection of samples
The peripheral blood of patients with CHB was collected, and PBMCs were isolated, treated with Trizol, and stored at −80°C. Liver enzymes ALT and AST were detected using an automatic biochemical instrument. HBV DNA and protein markers in serum were detected using qPCR and chemiluminescent immunoassay, respectively. Liver tissue samples were obtained by puncture biopsy, and hematoxylin and eosin (H&E) staining was performed to evaluate the degree of intrahepatic inflammation and fibrosis according to the liver pathological criteria in the "Guidelines for the Prevention and Treatment of Chronic Hepatitis B.”[15] chemerin, p-ERK, and NF-κB in liver tissue were detected by immunohistochemistry. The degree of inflammation was divided into Grade 0-4.The degree of fibrosis was divided into Stage 0-4.
Immunohistochemistry
Paraffin-embedded tissue sections were dewaxed to hydration and antigen repaired with a high-pressure. 3% hydrogen peroxide was added to the tissue for 15 min. Primary CMKLR1 and NF-κB antibodies (Proteintech, Wuhan, China) were incubated with tissues overnight. Streptomyces antibiotic peroxidase solution was added and incubated at room temperature for 20 minutes. The tissue sections were counterstained with hematoxylin and differentiated with hydrochloric acid ethanol. The stained tissues were imaged with a microscope(Leica DM2000, Leica Corp., German).
In vivo mouse model of liver injury
Concanavalin (Con) A(8ug/g weight)(Sigma Corp., MO, USA) was injected into the tail vein of 10 male BALB/c mice once a week for 3 weeks to induce liver injury. AgomiRNA-4804-3p(2 od/each mice) or an RNA negative control was injected into the caudal vein of mice. An orbital blood draw was collected before and 5 days after the second ConA injection. The levels of ALT and AST in blood were determined. The mice were euthanized by Carbon dioxide suffocation and the liver samples collected. Pathological changes in the mouse livers were stained with HE and imaged using a microscope(Leica DM2000, Leica Corp., German).
Statistical analysis
Data analysis was performed using SPSS17.0 statistical software (SPSS, Inc., Chicago, IL, USA). Data were expressed as mean ± standard deviation. The t test was used to compare the two groups. ANOVA and snk-q test was used for the multiple groups comparison. The data of non normal distribution are represented by quartile spacing. X2 test was used to compare the counting data. Pearson correlation was used to analyze. P < 0.05 was statistically significant.