Construction of NAFLD cell model
Human normal liver cells HL-7702 cells (Shanghai Institute of cell biology, Chinese Academy of Sciences) were seeded in 6-well plates at 5 × 105 cells/well. They were divided into control group and NAFLD model group (FFA group). After the adherent cells growth, cells in the control group were added with RPMI1640 medium containing 1% bovine serum albumin (BSA), and cells in the FFA group were added with high fat medium containing 1 mmol/L free fatty acid (FFA). Subsequently, cells were cultured in a 37 °C incubator containing 5% CO2. After induction of high-fat culture solution for 24 h, the cells were subjected to oil red O staining and triglyceride determination, respectively.
Cell transfection
HL-7702 cells were divided into control group (normal liver cells), FFA group (NAFLD cells), FFA + NC group (NARLD cells transfected with miR-499-5p negative-control inhibitor) and FFA + miR-499-5p inhibitor group (NAFLD cells transfected with miR-499-5p inhibitor). The control group was cultured in the usual medium, while the other three groups were cultured in the FFA high-fat medium. The normal liver cells and the successfully modeled HL-7702 cells were first collected, resuspended and counted, and then inoculated into a 24-well plate in an equal amount, and cultured in a 37°C and 5% CO2 incubator. When the cells were confluent to 60-80%, siRNA was transfected by liposome LipofectamineTM 2000, respectively. Next, 3 μL LipofectamineTM 2000 and 1 μL siRNA was diluted by 50 μL Opti-MEM® Reduced Serum Medium, and the liquid was kept at room temperature for 5 min after dilution. Then, the diluted siRNA and LipofectamineTM 2000 were gently mixed and incubated for 20 min at room temperature. Finally, miR-499-5p inhibitor and LipofectamineTM 2000 complex were added to each well, and the plates were gently shaken and incubated at 37 °C in a 5% CO2 incubator for 24 h.
Construction and grouping of NAFLD mouse models
Twenty-four SPF male C57BL/6 mice of 4 weeks old, weighing 18-20 g, were purchased from the Experimental Animal Center of Zhejiang Academy of Medical Sciences (Zhenjiang, China). Ordinary feed contained 8% rice bran, 51% corn, 30% soy flour, 3% bone meal, 1.3% multivitamin and 6.7% mineral, and high fat diet contained 80.5% basic feed, 2% cholesterol, 7% lard, 10% egg yolk powder and 0.5% bile salt.
After 7 days of adaptive feeding, 24 mice were randomly divided into SCD group and HFD group. Mice in SCD group (n = 8) were fed with basal diet, and mice in NAFLD model (HFD group) (n = 16) were fed with high fat diet. In addition, feeding environment conditions were a temperature range of 20-22 °C, a humidity range of 50-55%, and a brightness of 12 hours each. The experimental animals were free to eat and drink, and 4 was kept in one cage. After feeding for four weeks, 4 mice in the control group and the model group were sacrificed to test whether the NAFLD model was successfully constructed. Then, the NAFLD model group (n = 12) was subdivided into HFD group, HFD + NC group and HFD + miR-499-5p inhibitor group. The treatment was as follows: mice in the HFD group (n = 4) were injected with 100 μL physiological saline in the tail vein; lentiviral expression vector containing 100 mL unrelated sequence (viral quantity was 2 ´ 107 TU) was injected into caudal vein of mice in HFD + NC group (n = 4), and mice in the HFD + miR-499-5p inhibitor group (n = 4) were injected with a lentiviral expression vector containing 100 mL inhibitory sequence in the tail vein (viral quantity was 2 ´ 107 TU). Then, the high fat diet was continued for 4 weeks, and all mice were killed by decollation after 8 weeks. Before sacrificed, the mice were fasted overnight, the right eyeballs were removed and about 1.5 ml blood was taken for biochemical detection. Next, the middle left lobe of the liver was treated with HE staining for observation of pathological changes in the liver. Additionally, the remaining liver tissue was washed with PBS and preserved in liquid nitrogen. All animal experiments followed the guidelines for the management and use of laboratory animals.
Measurement of total cholesterol (TC) and aspartate aminotransferase (AST)
After feeding for 8 weeks, mice were fasted overnight and anesthetized by intraperitoneal injection of sodium pentobarbital (0.05 mg/g body weight). The eyeballs were removed and blood was collected with a 1.5 mL EP tube. The blood was centrifuged at 3000 rpm for 15 min at 4 °C to separate the serum. Finally, the levels of TC and AST in serum were measured using a Cobas8000 automatic biochemical analyzer (Roche, USA). After the blood collection, mice were decapitated and the liver was harvested.
qRT-PCR
Total RNA of normal liver cells HL-7702 and NAFLD cells was extracted by TRIZOL reagent (Thermo Fisher scientific, New York, USA). Simultaneously, total RNA of normal mouse liver tissues and liver tissues of NAFLD mice were extracted by RNAprep pure Tissue Kit (TIANGEN biotech Co., Ltd., Beijing, China). First, 1 μg total RNA was used as the initial template, and the total reaction system was 20 μL. The reverse transcription synthesis cDNA was performed on the Gene Amp PCR System 9700 using the FastQuant cDNA First Chain Synthesis Kit (FastQuant RT Kit (with gDNase), KR l06, TIANGEN, China). Subsequently, qRT-PCR was performed on a Rotor-Gene 3000 Real-time PCR instruments (Corbett Research) using a SuperReal Fluorescence Quantitative Premix Kit (SuperReal PreMix Plus, FP205, TIANGEN, China) and taking 1 μL cDNA as a template. The reaction conditions were pre-denaturation at 95 °C for 15 min, denaturation at 95 °C for 15 s, annealing at 60 °C for 60 s for 40 cycles, and U6 (F:5'-ATTGGAACGATACAGAGAAGATT-3'; R:5'-GGAACGCTTCACGAATTTG-3') as an internal reference for miR-499-5p (F:5'-ACTGCTTAAGACTTGGAGTGA-3'; R:5'-TACATTGGTGTCGTGGAGTCGGCAA-3'). The relative expression of miR-499-5p was calculated using the 2-∆∆Ct formula. The primers used in the experiment were synthesized by Invitrogen Company.
HE staining
Four mice were taken from each group, and they were sacrificed by cervical dislocation and soaked in 75% ethanol for 5 min. Next, thoracic liver tissue was taken and the blood was washed away by PBS. Then, liver tissue was paraffin-embedded, sectioned and stained with HE staining. The HE staining procedure was as follows: the sections were dewaxed by xylene and dehydrated by alcohol gradient, then stained with hematoxylin stain for 1 min, soaked in PBS for 1 min, rinsed with pure water until the sections were fully blue. Next, the sections were stained with eosin solution for 1 min, and then placed in gradient alcohol and xylene for dehydration and transparency. Finally, the neutral resin was mounted and the histopathological changes were observed under ordinary light microscope.
oil red O staining
The dry powder of 0.25 g oil red O was dissolved in isopropanol to 50 mL and stored at 4 °C in the dark. Before the cells and liver tissue were stained, 4 mL Oil Red O stock solution was diluted with 6 mL pure water to form Oil Red O staining solution. Immediately, the cell culture was removed and the cells were fixed with 10% neutral formaldehyde for 15 min. Then, the cells were stained with oil red O staining solution for 10 min and counterstained with hematoxylin for 5 min. After rinsing with ddH20, the cells were observed and photographed under the microscope.
The liver tissue stored in liquid nitrogen was taken out and cut into 5 μm thick at -34 °C. Then, the step of staining the liver tissue with Oil Red O staining solution was the same as the staining of the cells.
Determination of TG
TG was mainly determined by the GPO Trinder enzymatic method. The cell culture medium was aspirated, and 200 μL lysate was added to each well of a 6-well plate to lyse the cells. Next, the standard glycerin and cell lysate were mixed with the working solution, respectively, and the mixture was allowed to stand at 37 °C for 10 min. The TG concentration of the cells was measured at 570 nm using a microplate reader (Model 680, BIO-RAD, USA). Additionally, to detect the content of TG in the liver tissue of mice, 50 mg liver tissue was weighed, and then 1 mL of the lysate was added and ground into a uniform mixture. The remaining detection steps for the content of TG in mouse liver tissue were the same as procedure for cells.
Statistical analysis
Statistical analysis was performed using SPSS 19.0 (SPSS Inc., Chicago, IL, USA). The results were expressed as mean ± standard deviation (M ± SD). Statistical analysis was carried out using Student's t-test between two groups. All experiments were repeated at least three times. P < 0.05 indicated that the difference was statistically significant.