SUPPLEMENTARY MATERIALS AND METHODS
Human Tumor Samples
For this IRB approved study, tissues were obtained from patients undergoing surgical
resection and who were consented to the institution’s tissue collection protocol at Moffitt
Cancer Center between 2002-2012. Histopathological examination of specimens
(histological type, tumor size etc.) was performed by an experienced GI pathologist
(B.C.). Macrodissection of formalin-fixed paraffin-embedded (FFPE) tissues was
performed to isolate tumor sections (containing >80% tumor cells) for RNA extraction.
Microarray
RNA was extracted from FFPE material using the RecoverAllTM Total Nucleic Acid
Isolation kit optimized for FFPE samples (Ambion, Austin, TX) according to the
manufacturer’s protocol. All RNAs were quantified by using the Nanodrop ND-1000
spectrophotometer (Thermo Fisher Scientific, Pittsburgh, PA). Microarray analysis was
performed using the oligonucleotide probe-based Affymetrix U133 plus 2.0 chips per
standard Affymetrix protocols. Biotin-labeled cRNA generated by in vitro transcription
was fragmented and hybridized to the gene chip arrays at 45ºC for 16 hr and then washed
and stained with gene chip fluidics according to Affymetrix protocol. The probe arrays
were then scanned at 1.5-um resolution using the Affymetrix GeneChipScanner 3000.
Array Data Analysis
Scanned output files were visually inspected for hybridization artifacts and then analyzed
by using Affymetrix Microarray 5.0 software. Arrays were scaled to an average intensity
of 500 and analyzed independently. The MAS 5.0 software used for statistical algorithm
to determine the signal intensity of a transcript from the behavior of 11 different
oligonucleotide probes designed to detect the same gene. Probe sets that yield a change pvalue less than 0.006 was identified as changed. With replicate measurements the p-value
was adjusted. Alternatively, the signal intensity was calculated by the robust multi-array
analysis method (RMA) developed by Irizarry et al. Gene changes were then selected
using the fold-change cut-off available in RMA or using the Significance Analysis of
Microarrays (SAM) technique.
miRNA quantification
Total RNA was isolated from macrodissected FFPE tissues using the mirVANA™
miRNA Isolation Kit (Ambion, Austin, TX) according to the manufacturer’s protocol,
including enrichment for small RNAs. RNA concentration and quality were determined
using NanoDrop1000. Only RNA samples of sufficient quality were used.
Serum AFP quantification
Patient serum and growth media from cell lines were utilized for quantification of AFP in
the clinical chemistry laboratory at the Moffitt Cancer Center. Tumors were considered to
be AFP-secreting if the patient’s serum AFP level was above the upper limit of normal
(37IU) reported by our laboratory.
Quantitative RT –PCR Analysis
The differential expression level of mir-675 in HCC tumor samples and hepatoma cell
lines was confirmed by quantitative real-time PCR analysis. Total RNA was extracted
from tumor tissues and hepatoma cells. 1 ug of total RNA was reverse transcribed using
TaqMan reverse transcription reagent (Applied Biosystems, Carlsbad, CA). cDNA was
amplified in a 9700 Gene Amp PCR System (Applied Biosystems, Carlsbad, CA).
Using specific primers and probe for mir-675 (NCBI reference sequence NR_030533.1)
(Forward: 5’- AGG GTC TGG TGC GGA GA -3’ Reverse: 5’ GAG CGG TGA GGG
CAT ACA-3’ Probe: 5’-TCA CCA AGT CCA CTG TGG GC 3’). Experiments were
conducted in triplicate. Amplification reactions were run using 7900 HT real-time PCR
instrument (Applied Biosystems, Carlsbad, CA). Results were reported as the ratio
between the expression of mir-675 gene and the expression of Glyceraldehydes-3phosphate dehydrogenase.
Biological Pathway Analysis
MetaCore pathway analysis by GeneGo was used to identify the Gene GO pathways and
Gene GO networks for genes with statistically significant changes in expression, when
comparing transcriptomes of AFP-secreting and non-secreting human tumor samples as
well as miR-675 overexpressing and control hepatoma cells. Significance was calculated
using a built-in function of MetaCore software that utilizes a variation of the Fisher's
exact test adjusted for multiple sample testing with the Benjamini-Hochberg FDR
analysis.
MicroRNA Target Prediction
In silico microRNA miRNAMap 2.0 target prediction software was utilized to identify
potential targets of miR-675 (http://miRNAMap.mbc.nctu.edu.tw/). The DNA sequences
of the 3’-untranslated region (UTR) region of Twist1 and Rb mRNA was obtained from
Genbank of the National Center for Biotechnology Information webpage
(http://www.ncbi.nlm.nih.gov/).
Cell Culture and Transfection
The HepG2, SK-Hep and Hep3B cell lines were obtained from the ATCC. The cell lines
were authenticated in January 2012 by short tandem repeat (STR) analysis at the Genetic
Resources CoreFacility ay Johns Hopkins University using an Identifiler kit and
GeneMapper software (Applied Biosystems, Carlsbad, CA). Transfections were
performed in 6 well plates using Lipofectamine-2000 (Invitrogen, Carlsbad, CA) and 50
ng/2ml of pre-miR-675, negative control miRNA, anti-miR-675 or anti-miR control
(Applied Biosystems, Carlsbad, CA) according to the manufacturer’s protocol.
Proliferation
1 x 103 cell/ well were seeded in quadruplicate in 96-well plates. Cells were incubated for
72 hr, and cell proliferation was assayed using the XTT Cell Proliferation Kit II (Roche,
Mannheim, Germany). Proliferation was scored by measuring the spectrophotometrical
absorbance (wave length 540 nM) of the samples.
Cell Cycle Distribution assay
Analysis of cellular discrimination in Go/G1 versus S versus G2/M phases of the cell
cycle was conducted by measuring the cellular DNA content with propidium iodide
methodology. One million cells were washed in 1X PBS and transferred into tube
containing 70% ethanol. The cells were kept overnight at -20°C and fixed in 70% ethanol
and then rinsed with 1X PBS. The cells were then suspended in 1ml PI/Triton solution in
the presence RNase A. The cells were kept for 15 minutes at 37°C incubator. The cell
fluorescence was measured in the flow cytometer.
Western blot Analysis
Cell lysis and protein extraction were achieved using the TRIZOLkit (Invitrogen,
Carlsbad, CA). Extracted proteins were dissolved in 1% SDS and concentrations were
measured using the Bio-Rad protein assay kit (Bio-Rad). Protein (50 to 100 ug) per
sample was loaded and fractioned by electrophoresis on SDS-PAGE gel. Proteins were
then transferred to a nitrocellulose membrane using Trans-Blot SD, Semi -Dry Transfer
Cell (Bio-Rad, Hercules, CA). The membranes were blocked with 5% non-fat dry milk
for 2 hr and washed with TBST solution. Primary antibodies (1:1000) were diluted into a
dilution buffer, comprised of 1X TBS, 0.01% Tween-20 with either 5% BSA or 5% nonfat dry milk. The membranes were hybridized at 4ºC overnight and then washed.
Membranes were then reacted with secondary antibodies (1:2000) for two hours, washed
and developed using a chemiluminescent kit (commercially available antibodies:
Amersham, Piscataway, NJ, Cell Signaling, Beverly, MA; Santa Cruz Biotechnology,
Santa Cruz, CA).
Soft Agar Assay
To make the bottom layer, a 3% Bacto agar (BD Biosciences, San Jose, CA) stock was
made in DIDW and sterilized in the autoclave. At 48°C, 40 mL of the growth medium
were mixed with 10 mL of the 3% agar. 2mL/well was plated into the wells of a 12-well
tissue culture plate and solidified at room temperature. For the cell layer, the cells were
counted using trypan blue dye and a hematocytometer. 3000 cells/well were prepared and
resuspended in separate tubes, in which agar medium were mixed with the cells. Oneand-a-half mL of cell-agar mixture were dispensed on top of the base layer. The plates
were then incubated for 3 weeks. Cell line used in triplicates for the analysis. In order to
develop the plates, 1mg/mL MTT was dissolved in the growth medium. 150ul of MTT
dye was added in each well and incubated overnight. The colonies were counted and
photographed at microscopic core facility.
Invasion
Invasive potential was measured using CytoSelect 24-well Cell Invasion Assay (Cell
Biolabs Inc, San Diego, CA). Cells that invaded the lower surface of the membrane were
stained with DAPI (Invitrogen, Carlsbad, CA) and counted per 100 high-power fields
with an inverted microscope. All experiments were run in duplicate and performed on
two separate occasions.
Luciferase Reporter Assay
The 3’UTR clone of Twist1 (NM_000474) was purchased from Origene, Rockville, MD.
The pmiR target vector containing Twist1 3’UTR (Origene, Rockville, MD) was
transformed into competent cells (DH5α) and grown. Plasmid DNA was extracted and
measured. The restriction digest was performed with SgfI and MluI as recommended by
the manufacturer and confirmed the product size of Twist1 3’UTR. The plasmid DNA
with Twist1 3’UTR was sequenced and validated with gene bank sequence. Pre-miR-675
and the Negative Control (Pre-miR) were purchased from Ambion, Austin, TX.
A total 250K human HepG2 cells were seeded in each well of a 6 well plate; as the cells
became 60-70% confluent, the transfection assay was performed. There are 3 triplicates
for control and 3 triplicates for case. 1000ng of plasmid DNA and 50nM of pre-miR or
control miRNA were co-transfected into HepG2 cells for 48 hours. Each sample was also
cotransfected with 0.5ug of pRL-CMV plasmid-expressing Renilla luciferase to monitor
the transfection efficiency. At 48h post transfection, the activity of firefly luciferase was
measured by using the dual-luciferase reporter assay system as described by the
manufacturer (Promega, San Louis Obispo, CA). Relative luciferase activity was
normalized with Renilla luciferase activity.
Statistical Analyses
Determination of biological pathway dependence was undertaken using the basic formula
for hypergeometric distribution, with consideration given to the number of differentially
regulated genes, the number of genes in a given pathway, and the number of genes in all
pathways. Results of individual experiments were analyzed using 2-sided Student's t-test
with significance accepted with > 95% confidence. For microarray analysis, refer to
section 2.3.