SUPPLEMENTARY MATERIALS AND METHODS
Ethics statement
All experimental procedures were approved by the Institutional Review Board of the
Fourth Military Medical University. Written informed consent was obtained for all
patient samples. Animal experiments were performed with the approval of the
Institutional Committee for Animal Research, in conformity with national guidelines
for the care and use of laboratory animals.
Tissue microarrays and Immunohistochemistry
CRC tissue microarrays (shBiochip, Shanghai, China) included 90 cases of CRC
samples and the corresponding adjacent liver tissues with detailed clinicopathological
and follow-up information(Supplementary Table S1). The tissue microarray was
stained for STIM1 (Abcam, Cambridge, UK) expression. The array was scored
independently by two pathologists for both the staining intensity and the extent of
STIM1 expression across the section.
Immunohistochemical staining was performed using the the avidin-biotin complex
immunoperoxidase method. Pathologists rated STIM1 expression according to the
ratio of positive cells per specimen and the staining intensity. The ratio of positive
cells per specimen was evaluated as follows: 0 for ≤1% staining, 1 for 2 to 25%
staining, 2 for 26 to 50% staining, 3 for 51 to 75% staining, and 4 for ≥75% staining.
The staining intensity was evaluated as follows: 0, no signal; 1, weak; 2, moderate;
and 3, strong staining. Combined scoring was then calculated and graded as negative
(–; 0–1), weak (+; 2–4), moderate (++; 5–8), and strong (+++; 9–12).
Immunofluorescence staining
All cells were plated on glass-bottom dishes at approximately 30% confluence and
were allowed to grow for 24 h. Then, cells were fixed with 3.7% formaldehyde
solution for 15 min at room temperature, permeabilized with 0.1% Triton X-100 in
PBS for 3 min, and then blocked with 1% BSA in PBS for 1 h. Anti-E-cadherin or
anti-vimentin monoclonal antibody was incubated with blocked cells for 2 h. After
washing with PBS, cells were incubated with secondary antibody for 1 h，samples
were mounted with DAPI (Vector Laboratories, Burlingame, CA) according to the
manufacturer’s protocol. The Immunostained cells were detected and visualized ,
images were captured by a scanning confocal microscope(FlUOVIEW FVLIOi,
OLYMPUS).
RNA extraction and real-time quantitative reverse transcription polymerase
chain reaction
Total RNA was extracted from CRC cells and tissues using TRIzol reagent. For the
detection of miR-185 expression, stem-loop RT-PCR was performed using an
All-in-One™ miRNA qRT-PCR Detection Kit (GeneCopoeia, Rockville, MD, USA)
according to the manufacturer’s instructions. Briefly, the extracted RNA was
reverse-transcribed in the presence of a poly-A polymerase with an oligo-dT adaptor.
Real-time PCR was performed with SYBR green buffer, a forward primer for the
mature miRNA sequence, and a universal adaptor reverse primer. The relative
expression levels of miR-185 were calculated using the comparative threshold cycle
(CT) method and were then normalized to the expression levels of U6 small RNA .
The primers used for the detection of miR-185 and U6 were purchased from
GeneCopoeia (HmiRQP0247, HmiRQP9001).
To assess the mRNA expression levels of STIM1, qPCR was performed using a
TaKaRa
SYBR
Green
PCR
Kit
(TaKaRa).
Glyceraldehyde
3-phosphate
dehydrogenase (GAPDH) or beta-actin was used to normalize the mRNA expression
levels.
The
following
primers
were
used:
STIM1,
5'-GAATTGACAAGCCCCTGTGT-3'
(sense)
and
5'-ATGACTTCCATGCCTTCCAC-3'
(anti-sense);
GAPDH,
5'-CCGGGAAACTGTGGCGTGATGG-3'
5'-AGGTGGAGGAGTGGGTGTCGCTGTT-3'
(sense)
(anti-sense).
and
beta-actin,
5'-CTCCATCCTGGCCTCGCTGT-3' (sense); 5'-GCTGTCACCTTCACCGTTCC-3'
(anti-sense).
STIM2,
5'-ATGTCACTGAGTCCACCATGCTTTA-3' (sense);
5'-TCTCTGTGCAGATGGCTGTGTTTA-3'
(anti-sense).
ORAI1,
5′-CGTATCTAGAATGCATCCGGAGCC-3′(sense);
5′-CAGCCACTATGCCTAGGTCGACTAGC-3′(anti-sense).
ORAI2,
5'-GGGTCTTCGTGTCACAGCTTCA-3'
(sense);
5'-GCACTCCTGCAGAGCACACTTTAG-3'
(anti-sense).
5'-ACGCTATGCACCAAACTTGTTCTC-3'
ORAI3,
(sense);
5'-CCCAACCCTTGCTAAGGACTCA(anti-sense)
All reactions were performed in triplicate.
In vitro migration and invasion assay
A 24-well transwell plate (8 mm pore size; Corning, NY) was used for this assay.
For migration assays, the cells were added to the top chamber lined with a non-coated
membrane. For invasion assays, the chamber inserts were coated with 200 mg/ml
Matrigel (BD Biosciences, San Jose, CA) and dried overnight under sterile conditions.
The cells were then placed in the top chamber. In both assays, the cells were
suspended in medium without either serum or growth factors, and medium
supplemented with serum was used as a chemoattractant in the lower chamber. After
incubation at 37℃ in 5% CO2 for 24 h, the top chambers were swabbed to remove
residual cells. Invading cells on the undersides of the membranes were fixed in 100%
methanol for 10 min, air-dried, stained with 0.1% crystal violet and quantified with a
microscope. Five random fields were analyzed for each insert. The assays were
conducted in triplicate in three independent experiments.
Tail vein metastatic assay
To produce experimental metastasis, cells were washed and resuspended in PBS.
Five-week-old BALB/C-nu/nu nude mice obtained from the Shanghai Laboratory
Animal Center of China were injected into the lateral tail vein, and the animals were
maintained in a sterile animal facility. Each tumor cell subline was injected into ten
mice. After ten weeks, the mice were killed, and the lungs and liver were examined
for metastases. Tumor tissues derived from various organs were dissected and
examined histologically. The experiments were repeated two to three times.
Western blotting analysis
Cell proteins were extracted and separated with SDS-PAGE gels, and western blot
analysis was performed according to standard procedures. β-actin expression was
assessed as a loading control. Anti-STIM1 (Abcam, Cambridge, MA), anti-E-cadherin,
anti-Vimentin and anti-β-actin (Santa Cruz, CA) antibodies were used for the western
blot experiments.
Measurement of intracellular Ca2+
Cells seeded on coverslips were loaded with 3μM Fura 2-AM by incubation in
Hank’s balanced salt solution (136.9mM NaCl, 4.2mM NaHCO3, 0.3mM Na2HPO4,
5.4mM KCl, 0.4mM KH2PO4, 0.5mM MgCl2, 0.4mM MgSO4, 5.5mM D-Glucose,
1.3mM CaCl2, 8.0mM Hepes, pH7.2) for 30 min in the dark at room temperature,
followed by washing and an additional 30 min incubation to ensure full
de-esterification. Coverslips were then transferred to a perfusion chamber. For every
coverslip, at least 10 cells were measured simultaneously (excited at 340 and 380nm
with emission at 510 nm) using a ×20 objective xenon lamp (Lambda DG4, Sutter
Instrument Company, Novato, CA, USA) equipped with a Nikon epifluorescence
microscope (TE2000-U; Nikon, Tokyo, Japan) and band-pass filters for wavelengths
of 340 and 380 nm. Based on the equation: [Ca2+]c=Kd×(Sf2/Sb2) ×(R-Rmin)/(Rmax-R),
the [Ca2+]c can be represented by the ratio
(R) of 340/380nm fluorescence images. Cells were first perfused with
Hank’s balanced salt solution containing 1.3mM CaCl2 to determine the
resting cytosolic free Ca2+ concentration ([Ca2+]c). Then, the buffer was
replaced by Ca2+-free and 2mM ethylene glycol tetraacetic acid-containing Hank’s
balanced salt solution to remove extracellular Ca2+ . Once the 340 nm/380nm ratio
reached a steady state, Thapsigargin(TG) was added to achieve final 10 mM
concentration and Ca2+ released from ER ([Ca2+]ER) was monitored. When ER Ca2+
was depleted, 1.3mM CaCl2 was added back into the perfusate to record Ca2+ influx
(SOCE).
Statistical analysis
The data are presented as the means ± standard errors of the mean (SEM) or
medians (ranges). Student’s t-test (two-tailed) or a one-way ANOVA test was
employed to analyze the in vitro and in vivo data unless otherwise indicated (χ2 test).
The non-parametric Mann-Whitney test was used to analyze the relationship between
STIM1 or miR-185 expression levels and various clinicopathologic characteristics.
The survival rates were determined using the Kaplan–Meier method. Statistical tests
were performed using SPSS 17.0 software (Chicago, IL, USA). P < 0.05 was defined
as statistically significant. * P < 0.05; **P < 0.01.