1. A mouse model of NAFLD
We adopted 72 8-week-old C57 BL/6J mice, which were provided by Laboratory Animal Center of Chinese Academy of Sciences (Shanghai, China). Since the high fat diet is more likely cause TG accumulation in liver [24], we used the Western-type diet (containing 0.15% cholesterol and 21% fat) for feeding mice for 6 weeks under light/dark cycle leading to circadian rhythm sleep disorder in mice [25]. All mice were randomly divided into three groups: (1) mice that were subjected to normal diet and regular light/dark cycle (C57 ND group); (2) mice that received high-fat diet under normal light/dark cycle (C57 WD group); (3) mice that received high-fat diet under abnormal light/dark cycle (C57 WD + DD/LD group).
2. Cultivation Of Primary Hepatocytes
Primary hepatocytes were isolated from C57 ND mice in our vitro experiments. The cells were collected according to the collagenase perfusion method [26]. Arterial blood of the mouse liver was blocked and replaced by 5 ml/min EDTA solution (including 5 mM glucose, 0.5 mM EDTA, 4.15 mM NaHCO3, 136 mM NaCl, 5.4 mM KCl, 0.65 mM NaH2PO4, 0.85 mM Na2HPO4, and 1 mM HEPES) for 6 min. Then, the liver was perfused with 1 mg/ml collagenase type IV. The liver was isolated and cut into pieces. Tissue fragments were filtered, centrifuged, and washed. The cells were collected and incubated in William’s E medium, containing 10% fetal bovine serum (FBS). After 4 h, the old medium was replaced with a new William’s E medium, containing 10% FBS to remove the dead cells. These cells at passage 0 were directly utilized herein.
3. Short Interfering RNA (SIRNA) Transfection
The Rev-erbα siRNA was synthesized at RiboBio Co., Ltd. (Guangzhou, China). Besides, 100 nM siRNA (Rev-erbα siRNA or Control siRNA) and 50 nM LipofectamineTM 3000 (Thermo Fisher Scientific, Waltham, MA, USA) were incubated at room temperature for 5 min. The Rev-erbα siRNA or Control siRNA were mixed with LipofectamineTM 3000 for 15 min to promote the formation of Rev-erbα siRNA/Lipofectamine complex and Control siRNA/Lipofectamine complex. Then, these complexes were added into the serum-free William’s E medium, incubated for 6 h. Replace with a fresh William’s E medium for another 48 h. The interference was detected via quantitative reverse transcription polymerase chain reaction (RT-qPCR).
4. Histological Analysis
Histological examination was performed to assess fatty liver degeneration in mice. The liver samples were fixed in 1% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 24 h. Then, the samples were embedded into paraffin and processed routinely. Finally, the samples were stained with hematoxylin and eosin (H&E) as described previously [27].
5. RNA Extraction And Reverse Transcription
Total RNA was extracted from liver tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions. RNA concentration was determined by UV spectrophotometry. RNA integrity was assessed using agarose gel electrophoresis. Then, 1 µg RNA was reversely transcribed into cDNA using ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan) according to manufacturer’s protocol.
6. RT-QPCR
The cDNA was amplified by ABI QuantStudio5 (Applied Biosystems, Foster City, CA, USA). The qPCR reaction mixture included 2.5 µl cDNA, 1 µl of 100 nM upstream primers, 1 µl of 100 nM downstream primers, 10 µl SYBR Green Master mix (Bio-Rad Laboratories, Hercules, CA, USA), and 20 µl H2O. The relative expression levels of target genes were calculated using the 2ΔΔCt method and normalized to 18S. The qPCR primers are presented in Table 1.
Table 1
Gene names
|
Primers
|
Primer sequences (5’-3’)
|
Real time-PCR
|
|
|
Bmal1
|
Sense
|
CACTGACTACCAAGAAAGTATG
|
|
Antisense
|
ATCCATCTGCTGCCCTGAGA
|
Clock
|
Sense
|
ATGAGCACCAAGACCATTCC
|
|
Antisense
|
GCTTCAGTGCTCCCAACTTC
|
Per2
|
Sense
|
CAGACTCATGATGACAGAGG
|
|
Antisense
|
GAGATGTACAGGATCTTCCC
|
Cry2
|
Sense
|
GCTAGAGTGACGGAGATGCC
|
|
Antisense
|
ACTACCACCTCACTGGGACA
|
ULK1
|
Sense
|
TGGGGAGAAGGTGTGTA
|
|
Antisense
|
TACTCTACAACAAGGGGCACA
|
LC3Ⅱ
|
Sense
|
CCCAGTGATTATAGAGCGATACAAGGGGGAG
|
|
Antisense
|
CTGCAAGCGCCGTCTGATTATCTTGATGAG
|
Atg5
|
Sense
|
GGCACACCCCTGAAATGGCATTATCC
|
|
Antisense
|
CCTCAACCGCATCCTTGGATGGAC
|
Lamp1
|
Sense
|
GCCCTGGAATTGCAGTTTGG
|
|
Antisense
|
TGCTGAATGTGGGCACTAGG
|
Rev-erb α
|
Sense
|
TACATTGGCTCTAGTGGCTCC
|
|
Antisense
|
CAGTAGGTGATGGTGGGAAGTA
|
GAPDH
|
Sense
|
ACAGCCGCATCTTCTTGTGCAGTA
|
|
Antisense
|
GGCCTTGACTGTGCCGTGAATTT
|
ChIP
|
|
|
Arbp
|
Sense
|
GAGGTGGCTTTGAACCAGAG
|
|
Antisense
|
TCTTTGTCTCTGTCTCGGAAAA
|
Bmal1
|
Sense
|
GGAAAGTAGGTTAGTGGTGCGAC
|
|
Antisense
|
AAGTCCGGCGCGGGTAAACAGG
|
ULK1
|
Sense
|
GCCTGGACTACAGGAAACCC
|
|
Antisense
|
AACTGGCTGGCTTCAGACTC
|
Atg5
|
Sense
|
GCTGCTGACAGAGCAAAGTG
|
|
Antisense
|
GGGTTTGAGACAAGCTCTCG
|
RNA interfering
|
|
|
mRev-erbα siRNA
|
Sense
|
CUUCGUUGUUCAACGUGAATT
|
|
Antisense
|
UUCACGUUGAACAACGAAGTT
|
7. Chromatin Immunoprecipitation (CHIP) Assay
The ChIP assay was conducted using an EZ-ChIP™ kit (Millipore, Billerica, MA, USA) according to manufacturer’s instructions. The liver tissue was collected at ZT8, cut into 1 ~ 3 mm3 pieces, and moved into a 50 ml centrifuge tube. Cross-link proteins to DNA by adding 27 µL of 37% formaldehyde to the sample and incubate for 15 min at room temperature with shaking. Add glycine to a final concentration of 125 mM to the medium and incubate with shaking for 5 min at room temperature. Rinse cells twice with 10 mL cold phosphate-buffered saline (PBS). Add 5 mL of cold PBS, scrape dishes thoroughly with a cell scraper and transfer into 50 mL tube. Add 3 mL PBS to dishes, scrape again and transfer the remainder of the cells to the 50 mL tube. Centrifuge at 4 °C for 5 min at 1,000 rpm. Carefully aspirate off supernatant and resuspend the pellet in ChIP lysis buffer, and incubate for 10 min on ice. The chromatin solutions were precipitated with 5 µg Rev-erbα antibody (Cell Signaling Technology Inc., Danvers, MA, USA). Each reaction was performed at 4 ℃ for 12 h. Adopt Protein A Agarose to capture Rev-erbα complex. Elute the complex from Rev-erbα antibody and reverse protein-DNA crosslinks. The qPCR was performed to assess the enrichment of Rev-erbα protein.
8. Statistical Analysis
The data were presented as mean ± standard deviation (SD). Single cosinor method was employed to analyze circadian rhythm as previously described [28, 29]. The following equation was formulated for cosine function: Y(t) = M + A*cos(x*t + µ). The daily rhythm characteristics included mesor (midline estimating statistic of rhythm corresponding to the mean level), amplitude (half of the peak-to-trough difference of the fitted cosine function), and acrophase (the crest time of rhythm given in degrees (°C), where 360 °C is corresponding to a 24-h cycle) were estimated by the above-mentioned function. Differences between the values of each pair of parameters were compared by one-way analysis of variance (ANOVA), and P ≤ 0.05 was considered statistically significant.