Animals: Livers were dissected from C57Bl/6J mouse fetuses on day e16.5 and
e17.5 days of gestation. The day of observation of the copulation plug was day
e0.5. Livers were isolated from neonates on postnatal day 1. Adult liver was
obtained from 8-10 week old C57Bl/6J animals. All tissues were flash frozen in a
0
bath of isopentane over dry ice and stored at -80 C prior to RNA isolation.
Animals were housed in the AALAC accredited animal facilities in the Institute of
Animal Studies at Albert Einstein College of Medicine. Animals were treated
according to protocols approved by the Animal Care and Use Committee of the
Albert Einstein College of Medicine.
MicroRNA Gene Microarray
All oligos printed on the arrays had a 5' amino modifier at C6, and were diluted in
150 mM phosphate buffer, pH 8.0 or 3xSSC, to 50 µM for printing. Printing was
done under 50% humidity by the custom built microarray printer at the AECOM
Microarray facility (Aldo Massimi). For details of the equipment see
http://microarray1k.aecom.yu.edu/. Telechem SPH48 print head was used, with
pins spaced 4.5 mm center-to-center, populated with 16 split-tip tungsten pins
(Point Technology, Colorado), arranged in 4 x 4 array, each producing a 90 µm
diameter spot. Printed arrays were incubated at 70% humidity overnight.
miRNA microaray protocols.
Oligonucleotides were printed in quadriplicate on Corning Epoxide coated slides
(cat. 40042). The Genisphere Cy3 and Cy5 labeling method of probe preparation
(http://www.genisphere.com/) was used. Signal intensities were measured with a
Axon 4000 scanner. Data were processed using GenPix software (Axon, Garden
city, CA). Data analysis was performed using a computer script by Dr. Kate
Milova (http://microarray1k.aecom.yu.edu/bioinfo_tools/Rogler ).
Before hybridization, miRNA microarrays on glass slides were blocked according
to slide manufacturer (Corning) recommendations and hybridized using
Datascope/Genisphere Array 900 miRNA RT labeling Kit.
cDNA microarrays A list of cDNAs included on these arrays can be found at
http:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=glp1205 .
Total RNA was prepared from undifferentiated HBC-3 cells or cells differentiated
for 1-7 days with 3.5% DMSO as previously described (23). Cy3 and Cy5 labeled
targets were synthesized as previously described (26). Arrays were hybridized and
washed as described in (23).
Dual Luciferase Reporter Assays.
psiCheck2 miR16 was a gift of M. Landthaler (Rockefeller University), and it
contained an engineered miR-16 perfect match target site downstream of Renilla to
serve as a positive control. MiRidian Mimics and miRidian Inhibitors of miRs-23b.
27b, and 24 were obtained from Dharmacon. HEK293 cells were grown in 96 well
plates to ~30% confluence and transfected with 25 ng Reporter plasmid/well, plus
or minus Mimics or Inhibitors using Lipofectamine 2000 reagent according to
manufacturers instructions (Invitrogen). Mixtures of three mimics or inhibitors
contained a total of 0.75 pM/well. Media was removed and cells were lysed with
passive lysis buffer after 20 hours. 10 ul was used in the 96 well format for the
Dual luciferase reporter assays, according to manufacturers instructions (Promega)
in a BMG FLUOstar Optima. The Renilla/Firefly ratio for each experimental
plasmid treated with mimics was normalized against that plasmid alone. Each data
point is the average of quadruplicate measurements.
Primers used for psiCheck2 vector cloning.
Primers for cloning: Smad 3-3 forward primer, 5’-GTACCTCGAG
TAAGGCACCAGCCTGTTTCT-3’, reverse primer, 5,-CAGTACGCGGCCGC
GGGACACGGCTCTTTAACAA-3’; Smad 4-1 forward primer, 5,GTACCTCGAG GCCCTAACCATTTCCAGGAT-3’, reverse primer, 5’CAGTACGCGGCCGC TACTGCCACCTTGCAGAACA-3’; Smad 5-2 forward
primer, 5’-GTACCTCGAG GCTGTGAGCTGACATGGAAA-3’, reverse primer
5’-CAGTACGCGGCCGC TGGCACAGAAAACAAAAGGA.
In situ hybridization. Acetylated slides were treated with 5 ug/ml of proteinase K
(Boehringer) in PBS for 5 minutes and washed with PBS three times. Slides were
prehybridized in hybridization buffer (50% formamide , 5X SSC, 5X Denhardt’s,
200ug/ml yeast RNA, 500 ug/ml Salmon DNA, 20mg/ml blocking reagent
(Boehringerfor 4 hours at room temperature. Slides were hybridized with 20nM
digoxygenin labeled LNA microRNA (mir-23b or mir-122) Anti sense probes (Exiqon)
0
or control probe (C. elegans, mir-159 sense) for 20 hours at 55 C. Slides were washed
0
to a final stringency of 0.2X SSC, 60 C. Slides were washed in B1 (0.1M Tris pH 9.5,
0.1M sodium chloride), blocked in B1 containing 10% fetal bovine serum. Positive
hybridization was detected using anti DIG- alkaline phosphotase antibody diluted 1:200
in B1 containing 10%h fetal bovine serum and the alkaline phosphotase reaction was
developed using BCIP/NBT alkaline phosphate substrate (Vector) plus levamisol. Post
development the slides were washed with PBST, counterstained with nuclear fast red,
dehydrated and mounted with VectaMount (Vector).
Quantitative real time PCR was carried out using an Applied Biosciences gene
quantitation system SDS 7000. The oligonucleotides used as primers for liver gene
expression marker analysis are listed in Supplemental Methods. GAPDH was used
for normalization. The relative expression level of each gene was calculated using
-(∆∆Ct)
(28). The data presented are the mean of three replicates.
the formula 2
Primers used for QRT-PCR
Alb1-forward
Alb1-reverse
Apoc4-forward
Apoc4-reverse
Aqp1-forward
Aqp1-reverse
Cyp2c40forward
Cyp2c40reverse
Ggt1-forward
5'-CAGGTGTCAACCCCAACTCT-3'
5'-CCACACAAGGCAGTCTCTGA-3'
5'-ACCAGAACCAGGGACAGATG-3'
5'-CATAAAGCCCTGGACAGCTC-3'
5'-CATGAAGGTGTGGACCAGTG-3'
5'-ACCCTGGAGTTGATGTCGTC-3'
5'-CATCGATATGACCCCCAAAC-3'
5'-CATCTGGAAATTGGGAGGAA-3'
5'-AGGTTATCAATGCCCGTGAG-3'
Ggt1-reverse
H19-forward
H19-reverse
Hnf4a-forward
Hnf4a-reverse
Krt19-forward
Krt19-reverse
onecut1(Hnf6)forward
onecut1(Hnf6)reverse
Pck1-forward
Pck1-reverse
Smad3-forward
Smad3-reverse
Smad5-forward
Smad5-reverse
Ttr-forward
Ttr-reverse
5'-CCAGCTCATAACCACGGATT-3'
5'-CTCCTCCCCCTACCTTGAAC-3'
5'-CAGACATGAGCTGGGTAGCA-3'
5'-AGAGGTTCTGTCCCAGCAGA-3'
5'-ATGTACTTGGCCCACTCGAC-3'
5'-TTGAGACAGAACACGCCTTG-3'
5'-GGCTCTCAATCTGCATCTCC-3'
5'-TTCACACTTATGCGGGATGA-3'
5'-CCTTGCTGGGAGTTGTGAAT-3'
5'-CTGGCACCTCAGTGAAGACA-3'
5'-TCGATGCCTTCCCAGTAAAC-3'
5'-GCCTTTGTCTTCAGCACTCC-3'
5'-AGGCCAGCACAGGACTCTAA-3'
5'-TGCCTTTATGGGGAGTGAAG-3'
5'-CCACCCAAGTCCACAGAACT-3'
5'-TTTCACAGCCAACGACTCTG-3'
5'-TCTCTCAATTCTGGGGGTTG-3'
Quantitative RT PCR for miRNA.
Quantitative PCR for miRNA was carried out using miRVana QRTPCR primer
sets for mir-23b, mir-27b, and mir-24-1 (Applied Biosystem) according to the
manufacturers protocols. Mouse SnoRNA-292 probes were used as normalization
controls for the calculation of ∆Ct values.
Transfection of HBC-3 cells. HBC-3 cell were stripped of their feeder layer as
previously described (26) and replated at 40-60% confluence on plastic. The cells
were transfected using siImporter (Milipore) according to the manufacturer’s
protocol. miRNA inhibitors and mimics (Dharmacon) for mir-23b, mir-27b and
mir-24 and negative control mimics and inhibitors were used at a final
concentration of 10uM. SiRNA for Smad4 and scrambled siRNA control (IDT)
were used at a final concentration of 10uM. These conditions resulted in greater
than 95% of the cells transfected. 18 hours of post-transfection, cells were replated
5
2
at 1.0 -2.0 X 10 cells/cm in HBM (for undifferentiated cells), 3.5% DMSO in
HBM (to induce hepatocytic differentiation) or plated on Matrigel (to induce bile
duct morphogenesis) as previously described (23). Twenty four hours after
replating the cells were harvested for total RNA preparation using Qiagen
RNAeasy kits. Small RNA fractions were isolated as previously described (24).
Cells plated on Matrigel were photographed 24 hours after plating using a Nikon
Inverted phase microscope and digital camera with Spot software. 27 non
overlapping photographs were taken per treatment. Brightness and contrast of the
images were adjusted using Adobe Photoshop CS. The area of tubule and cluster
was quantitated using ImageJ software (NIH).
Northern Blot. 20 ug of total RNA isolated as described in (24) loaded per lane on
a 15% acrylamide TBE Urea gel (Invitrogen) and run at 180V for 60-75 minutes.
2ug of small RNA from adult mouse liver was loaded as a control. Antisense
32
oligonucleotide probes for individual miRNAs (IDT) were P end labled using T4
polynucleotide kinase. Hybridization was carried out using ULTRAhyb-Oligo
0
hybridization buffer (Ambion) overnight at 42 C. Blots were washed to a final
0
stringency of 1X SSC 42 C. Hybridization was imaged using a Molecular
Dynamics Phosphoimager and a STORM 860 Scanner (Molecular Dynamics).
RTPCR of mir-23b cluster. 1 ug Total RNA from undifferentiated HBC-3 cells
was reverse transcribed and RT-PCR for the mir-23b cluster was performed using
SuperScript One-Step RT-PCR (Invitrogen). Primers for the PCR reaction were:
genomic forward Xho I:5’- CTCGAGGGTGGCCTGGTGGATAGAC-3’;
genomic reverse Not I: 5’-GCGCCCGCCAGGCATTCTCACTGCTCAA-3’.
Transcription factor binding site mapping was performed using the Biobase
implementation of Transfac.