Further information can be found in Supplemental Methods.
Construction of the targeting vector and generation of the Mir122a–/– mice.
The BAC clone bMQ-418A13 (chr18: 65269984–65437465) containing the entire pri-Mir122a
locus was purchased from Geneservice. A genomic fragment of 13 kb encompassing 7.8 kb upstream and 5.1 kb downstream of pre-Mir122a
was cloned into PL253 in the bacterial strain EL350 by a recombineering-based method (19
). The genomic fragment of Mir122a
from PL253 was used to replace the WT allele of Mir122a
in 129/Sv mouse embryonic stem cells (MESCs). MESC clones containing the targeted allele were identified by Southern blot analysis. Several clones were isolated and transfected together with a vector encoding the Cre recombinase that allowed the deletion of a fragment of 1,544 bp containing the entire pre-Mir122a
. Clones with the Mir122a
knockout allele were identified by Southern blot analysis and were injected into C57BL/6J blastocysts. Germline transmission of the Mir122a–
allele was achieved by crossing the chimeric mice produced with normal C57BL/6 mice. Finally, homozygous Mir122a–/–
mice were generated by crossing heterozygous littermate offspring. The genotyping of the F1
and successive generations was performed by Southern blotting and by PCR (Supplemental Figure 1, B and C).
Serum biochemical studies, including analysis of total cholesterol, TG, ALT, and ALP, were performed monthly. Serum was collected and analyzed using a DRI-CHEM 3500S (Fujifilm).
Histology and immunohistochemistry.
Resected liver tissue was processed as either paraffin sections or cryosections. Oil red O staining was performed on frozen sections fixed with formalin. The paraffin sections were processed to allow H&E staining, periodic PAS staining, Sirius red staining, and immunohistochemical studies. The immunohistochemical analysis used antibodies against F4/80, CD31, collagen I (Abcam), PCNA, vimentin, MTTP, E-cadherin (Cell Signaling Technology), desmin (Millipore), and KLF6 (Abgent).
RNA isolation, gene expression, and high-density oligonucleotide microarray analysis.
Super RNApure (Genesis Biotech Inc.) was used to extract total RNA from the various frozen liver samples. Quantitative RT-qPCR was performed according to the manufacturer’s specifications with B2m
(β-2 microglobulin) RNA as an internal control (primer sequences in Supplemental Table 2). The microarray hybridizations were performed using total RNA prepared from the liver samples of 3 WT mice and 4 Mir122a–/–
mice at the age of 2 months; liver samples from 2 WT mice and tumor samples from 3 Mir122a–/–
mice at the age of 11 months; and liver samples from 3 WT mice and tumor samples from 5 Mir122a–/–
mice at the age of 14 months. GeneChip Mouse Genome 430 2.0 Affymetrix oligonucleotide Gene Chips (Affymetrix) were analyzed at the Microarray & Gene Expression Analysis Core Facility (VYM Genome Research Center, National Yang-Ming University) according to the Affymetrix protocols. All the data files are presented in compliance with MIAMI guidelines and can be accessed online at the Gene Expression Omnibus (GSE27713 and GSE31453). Microarray datasets were analyzed using GSEA (version 3.2) (37
) from the Broad Institute. Detailed information can be found in Supplemental Methods.
Blood for mouse serum lipoprotein analysis was obtained after 2 consecutive overnights (16 hours) of fasting. Serum lipoproteins were analyzed on the Hydragel K20 Electrophoresis System (Sebia) according to the manufacturer’s methodology.
Extraction of total lipids from liver.
Mice were fasted for 2 consecutive overnights (16 hours) before liver tissue sampling. A 0.2- to 0.5-g portion of the liver was frozen in lipid nitrogen and ground into a powder in a mortar. A 4-ml mixture of chloroform and methanol was added to create a suspension to allow the extraction of lipids (57
). The procedure was repeated twice. A total of 12 ml of extraction solution was used. The mixtures containing the extracted lipids were pooled into a 20-ml saponification tube. After adding 3 ml distilled water to the mortar in order to resuspend the tissue material, the resulting suspension was added to the extracts. The pooled suspension was then extensively vortexed (30 seconds 4 times, followed by centrifugation at 1,250 g
for 30 minutes. A 4-ml portion of the top layer and a 5-ml portion of the bottom layer were separately collected into 20-ml counting vials. The organic (bottom) layer was dried under a stream of N2
gas. The upper aqueous layer was concentrated on a centrifugal concentrator. The two residues were then stored at –80°C before NMR measurement.
The lipid residues were resuspended in 400 μl deuterated chloroform (CDCl3
). The solution was transferred to a 5-mm NMR tube. NMR measurements were carried out on a 400-MHz Fourier transform NMR (FT-NMR) spectrometer (Bruker) with a BDI probehead. The pulse sequence and data acquisition for the NMR measurements were similar to those reported by Beckonert et al. (22
). A reference sample containing 2 mg cholesterol in CDCl3
and under the same NMR conditions was used for comparison and quantification (signal intensity of H-18, chemical shift 0.65 ppm).
Identifying miR-122 targets among the upregulated genes of Mir122a–/– livers.
Three computational tools, namely miRanda (58
), TargetScanS (59
), and RNAhybrid (60
), which have been successfully integrated by us in miRNAMap previously (61
), were used in this study. In order to achieve higher prediction accuracy, we also integrated another tool, PITA (62
). The integrated tools were then used to identify the miR-122a target sites located within the accessible regions of the 3′ UTRs of the upregulated genes in the Mir122a–/–
mouse liver. Upregulated orthologous genes with target sites in both the mouse and human genomes were pinpointed. The analysis strategy and the performance evaluation are presented in Supplemental Methods and Supplemental Tables 6–8.
3′ UTR reporter assay.
The 3′ UTR fragments of the candidate target genes were subcloned using XhoI
downstream of the luciferase gene in the vector psi-CHECK2 (Promega). The negative controls were lenti-122M and lenti-GFP (15
). HEK293T cells were infected with lenti-GFP (293T-GFP), lenti-122 (293T-122), or lenti-122M (293T-122M) for 24 hours. Cells were then seeded into 24-well plates and co-transfected with 0.5 μg of the respective psi-CHECK2-3′ UTR construct using jetPEI (Polyplus Transfection). After 48 hours, luciferase activity was measured using the Dual-Luciferase Reporter Assay System Kit (Promega). The effect of miR-122a was expressed relative to the average value from 293T-122M cells. Three mutants of the miR-122a binding sites in the 3′ UTR of KLF6 were included in this study, namely KLF6-mu1, KLF6-mu2, and KLF6-mu1+mu2. The nucleotide sequences of all of the PCR cloning primers (Supplemental Table 9) and mutagenesis primers (Supplemental Table 10) are also listed.
Protein lysates (30 μg) were separated by electrophoresis on 10% SDS polyacrylamide gels and transferred onto PVDF membranes (Millipore) for immunoblotting. The membranes were incubated with primary antibodies overnight at 4°C and then with horseradish peroxidase–conjugated secondary antibody (PerkinElmer Life Sciences). Primary antibodies against apoB-100, apoB-48 (Novus), apoE, MTTP, vimentin, GAPDH, FASN, desmin, PTEN, phospho-Akt, Akt, phospho–c-Raf, c-Raf, phospho-MEK1/2, MEK1/2, phospho-Erk, Erk, phospho-GYS2, GYS2, E-cadherin (Cell Signaling Technology), and KLF6 (Santa Cruz Biotechnology Inc.) were used. Signals were detected by an enhanced chemiluminescence kit (PerkinElmer). The relative levels of protein expression were normalized against GAPDH.
A partial human pri-MIR122
gene was subcloned into the vector pcDNA3.1(B) (Invitrogen) and designated pcDNA-MIR122 (15
). Plasmid DNA was injected by the hydrodynamic technique as previously described (25
). Briefly, 20 μg of endotoxin-free plasmid DNA was dissolved in 2 ml of sterile pharmaceutical-grade saline at room temperature and injected into the mouse tail vein with a 26.5-gauge needle over 6 seconds. For the short-term study (1-month duration), all the mice received 2 injections, one on day 1 and one on day 15. For the long-term study (8-month duration), all the mice received one injection per month for the duration of the study. The WT mice were injected with the pcDNA3.1(B) HA vector DNA only, while the Mir122a–/–
mice were injected with either the pcDNA3.1(B) HA vector DNA or HA-miR-122 DNA. Each group included 5 or 7 mice that were 3 months old. Serum biochemical studies were carried out on day 5, day 14, or at the times indicated. The mice were sacrificed after 1 month or 8 months for histological examination and gene expression analysis. To restore the expression of MTTP in Mir122a–/–
mice, one dose of 20 μg endotoxin-free Mttp
expression construct (Origene MC204263) or pCMV6-NEO control vector (Origene) was delivered using the same injection protocol over 1 month. Each group included 5 mice that were 3 months old.
For delivery of shRNA expression constructs in vivo, all mice received a weekly dose of 20 μg of endotoxin-free shRNA construct using the same injection protocol for 2 weeks. Mice were sacrificed 14 days later. The shKlf6 (TRCN0000218241) and shLacZ (TRCN0000072224) constructs were obtained from TRC (National RNAi Core Facility, Academia Sinica). shLacZ targeting the β-galactosidase gene was used as a control for RNA interference. Each group consisted of 5 mice that were 7 months old.
Assays for serum TGF-β1.
Serum samples were frozen at –80°C until assayed. The serum levels of TGF-β1 were measured by a commercial ELISA kit (eBioscience) according to the manufacturer’s instructions.
All data are expressed as mean ± SD and were compared between groups using the 1-tailed Student’s t test. A P value of 0.05 or less was considered significant.
The animal studies were conducted in accordance with the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research and were approved by the IACUC of National Yang-Ming University.