MATERIALS AND METHODS
Cell lines and culture. HepG2, Huh-7, PLC/PRF-5, Hep-3B human HCC cells
were maintained in Dulbecco's modified Eagle's medium (DMEM) (GIBCO,
Gaithersburg, MD). Normal human hepatocytes (HH) were obtained from Sciencell (San
Diego, CA) and cultured following the manufacturer’s instructions.
Microarray analysis. RNA samples were used to synthesize double-stranded
cDNA using Superscript Double-Stranded cDNA Synthesis Kit (Invitrogen, Carlsbad,
CA). Double-Stranded cDNA was labeled and hybridized to 12x135K LncRNA
Expression microarray in NimbleGen Hybridization System including more than 23000
lncRNAs (Arraystar, Rockville, MD). Each transcript is represented by 1-5 probes to
improve statistical confidence. After hybridization and washing, the processed slides
were scanned with the Axon GenePix 4000B microarray scanner. Raw data were
extracted as pair files by NimbleScan software (version 2.5). NimbleScan software’s
implementation of RMA offers quantile normalization and background correction. The
Probe level files and Gene summary files were produced. The gene summary files were
imported into Agilent GeneSpring Software (version 10.0) for further analysis.
Differentially expressed genes and non-coding RNA were identified through Fold-change
screening.
Real-time PCR analysis. RNA was extracted using Trizol reagent and treated
with RNase-free DNase I (Qiagen, Valenica, CA). One μg RNA was reverse transcribed
to cDNA and quantitative real time PCR performed by SYBR Green. Expression of
MEG3 or coding protein genes was normalized to that of GAPDH. Expression of specific
mature miRNAs was assessed by using TaqMan human MicroRNA Assay Kit (Applied
Biosystem, Foster City, CA) and normalized to that of RNU6B (human) or RNU202
(mouse).
In situ RNA hybridization (ISH). A locked nucleic acid (LNA) probe with
complementarity to a 23bp section of MEG3 was labeled with 5′-digoxigenin and
synthesized by Exiqon (Woburn, MA, USA). Tissue sections on the TMA were digested
using 2 mg/mL pepsin and ISH performed as previously described (Nuovo et al., 2010).
Negative controls included omission of the probe and the use of a scrambled LNA probe.
Transfection. HepG2 and PLC/PRF-5 cells were transfected using the
Nucleofector system, solution V program T28 (Amaxa Biosystems, Koln, Germany)
following manufacturers’ protocol. siRNA control or anti-DNMTs (Dharmacon, Chicago,
IL), and miR control or miR-29a (Applied Biosystem, Foster City, CA) were used at the
final dose of 100 nM, while 1 ugr of MEG3-vector (Origene, Rockville, MD) or CMV6XL5 empty vector were used for each transfection.
Methylation Specific PCR (MSP). DNA was extracted by using phenolchloroform and treated with bisulfite using the EzDNA Methylation kit (Zymoresearch,
Orange, CA) according to manufacturer’s instruction. Bisulfite-treated DNA was then
used for methylation specific PCR (MSP) reaction as previously described (Benetatos et
al., 2010). Amplification was as follows: 95 ◦C for 15 min followed by five cycles at 94
◦C for 30 s, 70 ◦C for 30 s and 72 ◦C for 30 s; five cycles at 94 ◦C for 30 s, 65 ◦C for 30 s
and 72 ◦C for 30 s; 30 cycles at 94 ◦C for 30 s, 60 ◦C for 30 s and 72 ◦C for 30 s. Final
extension was performed at 72 ◦C for 7min. PCR products were identified by ethidium
bromide staining after 2% agarose gel electrophoresis.
Methylation sequencing analysis. Bisulfite-treated DNA was amplified by using
the following primers: F 5’-TTGATAGGAGAGATTGGATATTAGGTGTTTGG-3’, R
5’-CACCCCCTTTACAACCTATAAAACTTACCAAAAAC.-3’ The PCR product was
run in an agarose gel, the band cut and purified and cloned in Topo.TA.2 (Invitrogen) for
sequencing analysis.
Cell growth. For anchorage-dependent growth, cells were plated (1.5 x105 / well)
in 12-well plates. Trypan blue staining was performed at each time point and the number
of viable cells was expressed relative to cell counts at baseline. For anchorage
independent cell growth cells were seeded in 6-well plates (5000 per well) in appropriate
medium with 20% FBS after transfection. The final concentration of the agar system was
1.2% for the bottom layer and 0.8 % for the cell suspension layer. Anchorageindependent transformed cell growth and number of colonies was assessed after 2 weeks.
Colonies image were obtained with GelDoc Imagining instrument (BioRad, Hercules,
CA) and colonies number quantitated with the dedicated function of the QuantityOne
software (Biorad, Hercules, CA).
Generation of hepatocyte-specific miR-29a/b1 knock out mice. Liver-specific
miR-29a/b1 KO mouse (C57BL/6) was developed by serial breeding of miR29a/b1loxP/loxP and Albumin-Cre þ/þ (Alb-Cre þ/þ) mice. Genotyping was performed
by PCR in DNA extracted by tail. All experiments were reviewed and approved by the
Institutional Animal Care and Use Committee at the Ohio State University.
Cell cycle analysis and apoptosis assessment. Apoptotic cells were detected by
labeling DNA breaks through the Terminal Deoxynucleotide Transferase dUTP Nick End
Labeling (TUNEL) assay (Invitrogen, Carlsbad, CA). Cells were processed as indicated
by the manufacturer, stained by propidium iodide (PI) and Bromodeoxyuridine (BrDU),
and analyzed by flow cytometry with a BD FACScalibur (Heidelberg, Germany).
Reagents. 5-Aza-2-Deoxycytidine (5-Aza-dc) was obtained from Calbiochem
(Darmstadt, Germany) and diluted in dimethyl sulfoxide (DMSO). All reagents obtained
were of the highest purity available.
Statistical analysis. Results are expressed as mean ± SEM, unless indicated
otherwise. Comparisons between groups were performed using the two-tailed Student’s t
test. Regression analysis and scatter plot was performed by using the MedCal Software.
Significance was accepted when p was less than 0.05.
Western Blotting. Western blotting analysis was performed as previously
described (Braconi et al. 2010). The primary antibodies used were as follows: goat
polyclonal anti-DNMT1 (1:500; Santa Cruz Biotechnology), mouse monoclonal antiDNMT3b (1:500; Cell Signaling Biotechnology), and goat polyclonal anti-actin (1:500;
Santa Cruz Biotechnology).
SUPPLEMENTARY FIGURE LEGEND
Supplementary Figure 1: MEG3 expression was detected by RNA in situ
hybridization. Paraffin-embedded, formalin-fixed liver tissues were incubated with
locked nucleic acid probe anti-MEG3 with complementarity to a 23bp section of MEG3
and labeled with 5′-digoxigenin (Exiqon, Woburn, MA, USA). Tissue sections were
processed as previously described (Nuovo et al. 2010).
Supplementary Figure 2: Methylation status of the CpG dinucleotides in the
MEG3 promoter region. DNA from HepG2 cells or non malignant human hepatocytes
(HH) was bisulfite-treated and the MEG3 promoter amplified, cloned, and sequenced and
compared to non bisulfite-converted DNA. CpG dinucleotides are represented as short
vertical lines within the region. Seven different clones are represented for each sample.
Supplementary Figure 3: MSP was performed in DNA from human HCC and
matched adjacent tissues after bisulfite conversion. These cases were not matched with
those shown in Fig 1. The PCR product was separated by agarose gel electrophoresis and
bands were quantified using the Image J software (National Institute of Health). A
representative image and quantitative data of all samples are shown. For each sample, the
ratio between the intensity of the Unmethylated (U) and Methylated (M) band was
calculated and reported as the ratio between HCC and adjacent tissue.
Supplementary Figure 4: HCC cells were transfected with miR-29a. After 72
hours, protein was collected and analyzed by western blotting using antibodies for
DNMT3b (Cell Signaling, Danvers, MA), DNMT1 (SantaCruz, Santa Cruz, CA), and
actin (SantaCruz, Santa Cruz, CA).