Supplementary Materials and Methods
Tissue specimens and cell lines
Normal liver tissues were obtained from patients undergoing resection of hepatic
hemangiomas. HCC and adjacent nontumor liver tissues were collected from patients
undergoing resection of HCC. Both snap frozen tissues (in liquid nitrogen) and
formalin-fixed paraffin-embedded sections were obtained from the Bank of Tumor
Resources, Cancer Center, Sun Yat-sen University in Guangzhou, PR China. Both
tumor and non-tumor tissues were histologically confirmed. All patients were
unrelated ethnic Han Chinese who lived in Southeast China. The relevant
characteristics of the studied subjects are shown in Supplementary Table 1. No local
or systemic treatment had been conducted before operation, and no other anticancer
therapy was administered prior to relapse. Hepatitis B virus (HBV) or Hepatitis C
virus (HCV) infection was diagnosed when HBV surface antigen (HBsAg) or HCV
antibody (HCV-Ab) was detected by ELISA in the serum isolated from peripheral
blood. HBV and HCV infections were identified in 87.4% and 3.2% of HCC cases,
respectively. All HCC tumors originated from a background of chronic hepatitis or
cirrhosis. Informed consent was obtained from each patient and the study was
approved by the Institute Research Ethics Committee at the Cancer Center, Sun
Yat-sen University.
Cell lines that were used included HEK 293T and four human HCC cell lines (HepG2,
QGY-7703, MHCC97H and SMMC-7721). They were all maintained in Dulbecco's
modified Eagle's medium (DMEM, Hyclone, Logan, UT) supplemented with 10%
fetal bovine serum (FBS, PAA Laboratories GmbH, Pasching, Austria).
Northern blot, semiquantitative RT-PCR, real-time quantitative RT-PCR (qPCR) for
miRNA
For Northern blot, RNA was separated on a 15% denaturing polyacrylamide gel. The
loading buffer used contained 0.05% xylene cyanol FF and 0.05% bromophenol blue,
which comigrates with ~30 bp and ~10 bp RNA fragments in 15% denaturing
polyacrylamide gel, respectively. Primers used for RT-PCR and probes for Northern
blot are listed in Supplementary Table 2. RNA bands were quantified using GeneTools
software (version 3.03, SynGene, Cambridge, UK).
For qPCR, the reverse transcription was performed using miR-29b- or U6-specific
stem-loop primer, followed by real-time PCR with molecular beacon probes using
LightCycler® 480 (Roche Diagnostics, Germany). All reactions were run in duplicate.
The cycle threshold (Ct) values should not differ more than 0.5 between duplicates.
The miR-29b level was normalized to U6 expression.
Apoptosis analysis
Apoptosis was evaluated by morphological examination and the terminal
deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay. For
morphological examination, cells were stained with 4′-6′-diamidino-2-phenylindole
(DAPI; Sigma-Aldrich, St. Louis, MO) and those with fragmented or condensed
nuclei were counted as apoptotic cells. At least 500 cells were examined for each
sample. TUNEL staining was conducted using the DeadEnd fluorometric TUNEL
system (Promega), according to the manufacturer’s protocol. At least 400 cells were
counted for each sample.
Tumorigenicity assays in nude mice
All experimental procedures involving animals were performed in accordance with
the Guide for the Care and Use of Laboratory Animals (NIH publications Nos. 80–23,
revised 1996) and according to the institutional ethical guidelines for animal
experiments. miR-29b- and NC-transfected HepG2 cells (1106) were suspended in
100 μl PBS and then injected subcutaneously into either side of the posterior flank of
the same female BALB/c athymic nude mouse at 5-6 weeks of age. Six nude mice
were included and tumor growth was examined every three days over a course of 4
weeks. Tumor volume (V) was monitored by measuring the length (L) and width (W)
of the tumor with calipers and was calculated with the formula V = (LW2) 0.5.
Luciferase reporter assay
HEK293T or HepG2 cells plated in a 48-well plate were co-transfected with 5 nM
RNA duplex, 10 ng of firefly luciferase reporter comprising wild-type or mutant
3'-UTR of target gene, and 2 ng of pRL-TK (Promega). For the antagonism
experiment, HepG2 cells grown in a 48-well plate were first transfected with 200 nM
anti-miR-C or anti-miR-29, which was a mixture of anti-miR-29a, anti-miR-29b and
anti-miR-29c. Twenty-four hours later, the RNA-transfected HepG2 cells were
co-transfected with 10 ng of firefly luciferase reporter containing wild-type or mutant
3'-UTR of target gene and 2 ng of pRL-TK. Cells were collected 48 h after last
transfection and analyzed using Dual-Luciferase Reporter Assay System (Promega).
Luciferase activity was detected by M200 microplate fluorescence reader (Tecan,
Grodig, Austria). The pRL-TK vector that provided the constitutive expression of
Renilla luciferase was used as an internal control to correct the differences in both
transfection and harvest efficiencies. Transfections were done in duplicates and
repeated at least thrice in independent experiments.
Western blot
The sources of antibodies used for Western blot: rabbit polyclonal antibodies against
Mcl-1 (Santa Cruz Biotechnology, Santa Cruz, CA) or Bcl-2 (Cell Signaling
Technology, Beverly, MA); mouse monoclonal antibodies against cytochrome c
(Invitrogen, Carlsbad, CA) or β-actin (Boster, Wuhan, China). Protein bands were
quantified using GeneTools software (version 3.03, SynGene).
Immunohistochemisty (IHC)
Formalin-fixed, paraffin-embedded tissue was cut into 5 μm section, placed on
polylysine-coated slide, de-paraffinized in xylene, rehydrated through graded ethanol,
quenched for endogenous peroxidase activity in 0.3% hydrogen peroxide, and
processed for antigen retrieval by microwave heating in 10 mM citrate buffer (pH 6.0).
Sections were incubated at 4°C overnight with Bcl-2 or Mcl-1 mouse monoclonal
antibody (Santa Cruz Biotechnology). Immunostaining was performed using
ChemMate DAKO EnVision Detection Kit, Peroxidase/DAB, Rabbit/Mouse (code K
5007, DakoCytomation, Glostrup, Denmark), which resulted in a brown-colored
precipitate at the antigen site. Subsequently, sections were counterstained with
hematoxylin (Zymed Laboratories, South San Francisco, CA) and mounted in
non-aqueous mounting medium. All runs included a no primary antibody control.
Bcl-2 and Mcl-1 staining was evaluated under a light microscope at a magnification of
400×. For each specimen, ten images of representative areas were acquired and a total
of 1000 to 2000 tumor cells were manually counted. IHC scoring was performed
using modified Histo-score (H-score), which includes semiquantitative assessment of
both percentage of positive cells and intensity of staining. The intensity score was
defined as follows: 0, no staining; 1, weak staining; 2, moderate or strong staining.
The fraction score was based on the proportion of positively stained cells (0-100%).
The intensity and fraction scores were then multiplied to obtain H-score, which
ranged 0-2.