All animal experiments were approved and carried out in accordance with the Institutional Animal Care and Use Committee of the University of Ningxia Medical University. Eight to ten weeks old cystathionine beta-synthase (CBS) heterozygous knockout mice (cbs+/−) were obtained from Jackson Laboratory (Bar Harbor, ME) and maintained in the animal facility center at Ningxia Medical University. The mice were fed with regular diet plus 2.0% methionine chow and water ad libitum. Mice genotypes were determined by PCR.
Cell culture, infection and transfection
Human hepatocytes (HL-7702) were obtained from the Japanese Collection of Research Bioresources and cultured in RPMI-1640 medium (Thermo, USA) containing 10% fetal bovine serum (FBS) (Hyclone, USA) and 1% penicillin (Thermo, Waltham, MA, USA). The cells were infected with adenoviruses encoding PSMD10 (Ad-PSMD10) when they were 80% confluent. The control cells were infected with Ad-GFP. Hepatocytes were transfected with non-silencing small interfering RNA (NC, 5’-UUCUCCGAACGUGUCACGUTT-3’), PSMD10 siRNA (5'-GCCGGGAUGAGAUUGUAAATT-3'), GRP78 siRNA (5’-GGGCAA AGAUGUCAGGAAATT-3’), miR-212-5p mimics (5’-ACCUUGGCUCUAGACUGCUUA CU-3’) and miR-212-5p inhibitor (5’-AGUAAGCAGUCUAGAGCCAAGGU-3’) using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA).
Liver tissue preparation and morphologic observation
All mice were fasted but supplied with water for 24 h followed by peritoneally injection with 3% pentobarbital sodium in a dose of 2 mL/kg. Abdominal cavity of anesthetic mouse was incised, and then inferior vena cava blood was collected using 10 mL syringe. After standing for 3h at 4 °C, blood samples were centrifuged at 5000 × g for 15 min. Supernatant was stored at −80 °C until future use. Liver was incised and two segments (1.0 cm ×1.0 cm × 0.3 cm) were cut from right lobe of the liver. Two liver segments were fixed in 10 % neutral formalin and embedded in optimum cutting temperature compound (OCT). To observe morphologic changes in liver tissues, liver segments was HE stained.
Liver tissues were stained using a TUNEL Staining Kit (Roche Inc., Basel, Switzerland) and the TUNEL-positive cells were symbolized by fluorescein-dUTP with dNTP according to the manufacturer’s protocol of the in-suit apoptosis Detection Kit (Roche Inc, Basel, Switzerland). Cells with nuclear condensation/fragmentation and apoptotic bodies in the absence of cytoplasmic TUNEL reactivity (green staining of nuclei) were considered as apoptotic cells. Cell nuclei was counterstained with DAPI and visualized by fluorescent microscopy.
Western blot analysis was performed as previously described. Liver tissues and HL-7702 cells were lysed in a lysis buffer (KeyGEN, China) containing the protease inhibitor phenylmethanesulfonylfluoride (PMSF, KeyGEN, China) at 4 °C for 30 min followed by centrifugation to remove cell debris. Protein concentration was measured using BCA protein assay kit (Beyotime Institute of Biotechnology). The protein was separated by SDS-PAGE and transferred to PVDF membranes followed by examination with antibodies against PSMD10, cleaved caspase3, caspase12, Bax, Bcl2, PARP, ATF6, p-PERK, PERK, eif2a, p-eif2a, IRE1a, p-IRE1a, CHOP and β-actin (all from Abcam Inc., Cambridge, MA, USA) respectively. Signal intensity was analyzed with Bio-Rad image analysis (Bio-Rad, Hercules, CA, USA).
Real-time PCR (qRT-PCR)
Total RNA was isolated from livers or cultured cells using the miRNA isolation kit (Ambion), and reverse transcribed using TaKaRa Master Mix (Dalian, China). Real-time PCR examination of PSMD10, GAPDH, miR-212-5p and U6 were performed using Maxima SYBR Green/ROX qPCR Master Mix assay (Thermo Scientific). The expression levels of mRNA and miRNA were normalized using GAPDH or U6 as a reference gene. qRT-PCR primer sequences are listed in Table 1.
The paraffin embedded mouse liver tissue sections were permeabilized in PBS containing 0.1% Triton X-100 for 5min, and then incubated with blocking solution (5% goat serum in PBS) at room temperature for 30min, followed by incubation with primary antibody (PSMD10, 1:50, KDEL receptor, 1:50) overnight at 4 °C. After washing with PBS for 3 times, tissues were incubated with fluorescein conjugated secondary antibodies (goat anti-mouse or goat anti-rabbit) at RT for 1 h. After washing with PBS for 3 times and they were stained with DAPI for 5 min. Sections were then assessed using fluorescence microscopy (OLMPUS FV1000 confocal laser scanning microscope, Tokyo, Japan).
Live/Dead staining assay
Cell viability was monitored under laser scanning confocal microscope (LSM710) using cell viability Imaging Kit (Roche Diagnostics, 06432379001) according to the manufacturer’s instruction.
Hepatocytes apoptosis was detected using the FITC-Annexin V/PI staining kit. After homocysteine treatment, the cells were washed with ice-cold PBS, and incubated with fluoresce in conjugated Annexin V and PI for 15 min, cell apoptosis was analyzed using a FACS flow cytometer equipped with the FACS talion data management system and Cell Quest software (Becton Dickinson, San Jose, CA, USA).
Dual luciferase assays
Hepatocytes in 6-well plates were co-transfected with 200 ng of either pMIR-PSMD10-wt(5’-GAGAGTGGAAGAAGCAAAACTGCTGGTGTCCCAAGGAGCAAGTATTTACATTGAGAATAA-3’) or pMIR-PSMD10 mut (5’-GAGAGTGGAAGAA GCAAAACTGCTGGTGTCGGTTCCAGCAAGTATTTACATTGAGAATAA-3’) together with 100 nM of miR-212-5p mimics or non-target miRNA mimics using Lipofectamine 2000. In 48 h after transfection, firefly and renilla luciferase activities were measured using the Dual-Glo luciferase assay kit (Gibco Life Technologies).
Cells were lysed in a lysis buffer containing protease inhibitor on ice. After centrifugation, the supernatant was incubated with an anti-PSMD10, anti-GRP78 or normal rabbit IgG respectively at 4°C overnight followed by incubation with Dynabeads Protein G. Immunocomplex was separated by SDS-PAGE and proceeded for western blot analysis.
Results are expressed as the mean ± SD from at least three independent experiments. The data were analyzed using one-way ANOVA and additional analysis using the Student Newman-Keuls test for multiple comparisons within treatment groups or t-test for two groups. P<0.05 was considered to be statistically significant.