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Supporting Materials and Methods
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miRNA Microarray. One-color RNA labeling and hybridization on mammalian miRNA V1.0
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(CapitalBio) was performed to determine the miRNA expression pattern of HepG2.2.15 and HepG2 cells
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as described previously1. The human miRNAs that had an average signal intensity more than 1000 on
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each array present in over 50% of the arrays were selected for SAM analysis. The parameter settings were
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the same as for liver tissues with FDR threshold at 0 and fold change > 2 fold. Representative miRNAs
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were validated by the stem-loop real-time RT-PCR2.
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Constructs and siRNA Duplex. The miRNA minigenes were PCR-amplified from the human genomic
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DNA and cloned into pcDNA3.1 (Invitrogen) to generate pcDNA3.1/miRNA. PCR mutagenesis was used
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to produce mutant miRNA construct (pcDNA3.1/miRNAm). NFIB coding region was PCR-amplified
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from the HepG2 cDNA and cloned into pCMV-myc (Clontech, Mountain View, CA) to generate
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pCMV-myc/NFIB. The 3′-UTR of human NFIB fragment containing the two predicted target sites of
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miRs-372/373 was PCR-amplified from HepG2 cDNA and cloned into the Xba I site of pGL-3 control
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vector (Promega) to generate pGL/NFIB 3′UTR. Muta-directTM (SBS, Beijing, China) was used to
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produce mutant NFIB 3′UTR construct (pGL/NFIB 3′UTRm) in which both predicted sites for the seed
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region of miRs-372/373 were deleted. NFIB-specific siRNA duplex (siNFIB), negative control siRNA
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duplex (NCi) , and FAM-labeled NCi were synthesized by GenePharma (Shanghai, China). All constructs
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were sequence verified. The primers and siRNAs used are listed in Supporting Table 5.
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Cell Culture and Transfection. HepG2 and HepG2.2.15 cells were maintained as described previously3.
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Transfection was carried out by using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s
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protocols. Transfection efficiency was monitored using green fluorescent protein (GFP) expression
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plasmid or FAM-labelled NCi. HepG2 cells were transfected with pcDNA3.1/miRNA or empty vector
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and maintained in 1 mg/ml G418 selective medium for at least two weeks to generate stable
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miRNA-overexpressing or control cell lines. Overexpression of mature miRNAs was verified by
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quantitative real-time RT-PCR.
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HBsAg and HBeAg Assays. 1.3×HBV (10 ng) was cotransfected into HepG2 cells in 96-well plates
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together with 200 ng of constructs or 40 nM of siRNA duplex. 1.3×HBV (40 ng) and carrier pGEM-3Z
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plasmid (0.6 μg) were transfected into the stable miRNA-overexpressing HepG2 or mock cells in 24-well
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plates. Alternatively, 0.6 μg of NFIB construct or empty vector was transfected into HepG2.2.15 alone.
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The culture supernatants were collected and measured for HBsAg and HBeAg expression by the
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commercial ELISA assay kit (InTec).
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Purification of HBV Core Particle-associated DNA. 1.3×HBV (40 ng) was cotransfected into non-HBV
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producing cells in 24-well plates together with 0.6 μg of constructs or empty vector. Alternatively, 0.6 μg
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of NFIB construct or empty vector was transfected into HepG2.2.15 alone. Four hours after transfection,
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the growth medium was replaced by fresh medium containing either 0.1% DMSO or 4 μM lamivudine.
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The medium was replaced every 24 h for consecutive three days. HBV core particle-associated DNA was
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purified as described previously4.
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Luciferase Assay. 1.3×HBV-luciferase (10 ng) was cotransfected into cells in 96-well plates together
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with 200 ng of constructs or 40 nM of siRNA duplex. Alternatively, 10 ng of wild or mutant pGL/NFIB
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3′UTR or pGL-3 control vector was cotransfected into HepG2 cells together with 200 ng of miRNA
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constructs or empty vector. In each transfection, 1 ng of Renilla plasmid was used as the normalized
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control. Luciferase activity was measured by using the dual-luciferase reporter assay system (Promega).
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Nucleic Acid Isolation, Reverse transcription, Semi-quantitative PCR and Quantitative PCR. RNA was
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extracted from the cultured cells with TRIZOL reagent (Invitrogen), and RNA and DNA were isolated
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from the FFPE sections with the RecoverAll™ Total Nucleic Acid Isolation Kit (Ambion). A modified
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stem-loop RT-PCR reaction was used to quantify the mature miRNA2. Briefly, 100 ng of total RNA was
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reverse transcribed to cDNA using 50 nM of miRNA specific stem-loop RT primer. The 1/20 volume of
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RT product was used for PCR amplification (95°C for 5 min followed by cycles of 95°C for 30 s and
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60°C for 1 min). Some 28 amplification cycles were performed for semi-quantitative PCR, and 40 cycles
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were performed for quantitative real-time PCR (qPCR). U6 snRNA was used as the reference control. To
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determine the level of NFIB expression, cDNA was synthesized using Oligo(dT)15 primer (Promega).
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The qPCR was activated at 95°C for 5 min followed by 40 cycles of amplification (95°C for 15 s; 58°C
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for 20 s; 72°C for 20 s). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for endogenous
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control. To determine the level of HBV core particle-associated DNA, 1/50 volume of purifed DNA
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product was quantified by qPCR using the same amplification condition as NFIB. The 20 ng of isolated
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DNA was used for the detection of hepatic HBV DNA or miRs-371-3 gene copy number variation.
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Hemoglobin, beta (HBB) gene was used as the reference control. To effectively detect the DNA purified
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from FFPE tissues by using qPCR, The primers for HBV, miRs-371-3 and HBB genes were designed to
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produce short and non-redundant amplicons (90 bp for HBV and miRs-371-3, and 111 bp for HBB) with
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the annealing temperature of approximately 60°C using Primer 3 (http://frodo.wi.mit.edu/primer3/)5. The
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qPCR was performed using EvaGreen reagents (Biotium, Hayward, CA) on a RT-CyclerTM 436
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instrument (CapitalBio) or a 7900HT Fast Real-Time PCR System (Applied Biosystems). The relative
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expression of gene compared to reference control was calculated using the 2- ΔΔ Ct or - Δ Ct method. See
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Supporting Table 5 for details of RT and PCR primers.
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Western Blotting. Cell extracts were separated by SDS polyacrylamide gel electrophoresis, electroblotted
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to polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA), and blocked with TBS buffer plus 5%
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non-fat milk and 0.5% Tween-20. The membrane was then incubated with antibodies for NFIB (Sigma, St
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Louis, MO), myc-tag (TIANGEN, Beijing, China), or β-tubulin (Sigma) overnight at 4°C. After washing
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and incubating with HRP-linked secondary antibody (Cell Signaling, Danvers, MA), the bands were
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visualized by chemiluminescence (Pierce).
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Northern Blotting. Total RNA was isolated from cells using TRIZOL reagent (Invitrogen) according to
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the manufacturer’s protocol. The isolated RNA was digested with RNase-free DNase I and 20 μg of
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purified RNA was fractionated on a 1.2% formaldehyde agarose gel, transferred onto a Hybond N nylon
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membrane (Amersham, Little Chalfont, UK), fixed by baking for 30 min at 120 °C. The blot was
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prehybridized with DIG Easy Hyb (Roche, Basel, Switzerland) for 30 min at 50 °C and hybridized
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overnight with a DIG-labeled HBV probe. The probe was generated by PCR labeling of the 1.3×HBV S
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and X gene region using DIG DNA labeling reagent (Roche, Indianapolis, IN). After washing, the blot
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was detected by incubation with anti-digoxigenin-AP and luminescence CSPD (Roche) and exposed to
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X-ray film. See Supporting Table 6 for the primers used for probe labelling.
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mRNA Microarray. The mRNA expression profiles of two stable HepG2 clones overexpressing
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miRs-371-3 cluster versus mock HepG2 cells were performed by the human 22K Oligo microarray V2.0
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(CapitalBio) as previously described3. Genes with consistently more than 1.5 fold expression differences
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in two independent clones were selected. Clustering and visualization were performed by Cluster and
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TreeView. Allegro6 identified over-represented transcription factor (TF) binding motifs in promoters from
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-1000 to +200 bp on co-regulated genes using human whole genome (background) and a modified
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TRANSFAC Professional r8.2 database that excluded non-human TF binding motifs. Gene functional
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enrichment was performed by Functional Annotation tools in the Database for Annotation, Visualization,
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and Integrated Discovery (DAVID)7 using GOTERM_BP_ALL module with count threshold ≥ 2 & EASE
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< 0.05.
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Supporting References
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1.
Guo Y, Chen Z, Zhang L, Zhou F, Shi S, Feng X, Li B, et al. Distinctive microRNA profiles relating
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to patient survival in esophageal squamous cell carcinoma. Cancer Res 2008;68:26-33.
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quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 2005;33:e179.
Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, et al. Real-time
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3.
Guo Y, Guo H, Zhang L, Xie H, Zhao X, Wang F, Li Z, et al. Genomic analysis of anti-hepatitis B
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virus (HBV) activity by small interfering RNA and lamivudine in stable HBV-producing cells. J Virol
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2005;79:14392-14403.
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4.
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replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones.
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Gastroenterology 2006;130:823-837.
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5.
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Methods Mol Biol 2000;132:365-386.
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6.
Pollicino T, Belloni L, Raffa G, Pediconi N, Squadrito G, Raimondo G, Levrero M. Hepatitis B virus
Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers.
Halperin Y, Linhart C, Ulitsky I, Shamir R. Allegro: analyzing expression and sequence in concert to
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discover regulatory programs. Nucleic Acids Res 2009;37:1566-1579.
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7.
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using DAVID bioinformatics resources. Nat Protoc 2009;4:44-57.
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Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists