MiR-106b induces cell radioresistance via the PTEN/PI3K/AKT pathways and p21 in colorectal cancer

Clinical specimens

Colorectal cancer tissues were collected from fresh surgical specimens, frozen in
liquid nitrogen, and stored at ?80°C until further analysis. All tissues had been
histologically confirmed to be adenocarcinoma. Pathologic verification and classification
were performed based on the system of the International Union Against Cancer. The
research protocol was approved by the Ethics Committee at Nanfang Hospital, and written
consent was obtained from all patients for the use of their tissues.

Cell culture

Human colonic carcinoma cell lines HT29, SW480, SW620 and LOVO were obtained from
American Type Culture Collection (ATCC). The CRC cell lines were cultured in RPMI-1640
medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA), 100 IU/ml
penicillin and 100 g/ml streptomycin in humidified 5% CO2 at 37°C.

RNA isolation, reverse transcription, and qRT-PCR

The total RNA was extracted from CRC cell lines with RNAiso Plus (Takara, Japan).
To detect miR-106b, a stem-loop reverse transcription-polymerase chain reaction (RT-PCR)
was performed using All-in-One TM miRNA quantitative RT-PCR (qRT-PCR). The sequence-specific
forward primers for mature miR-106b and the U6 internal control were 5-TGTAAAGTGCTGACAGTGCA-3
and 5-CTCGCTTCGGCAGCACA-3, respectively. The relative expression was calculated via
the comparative cycle threshold (Ct) method using the expression of U6 small nuclear
RNA as the reference. The qRT-PCR for the analysis of mRNA expression was performed
on a Stratagene ABI PRISM7500 Fast Real-time PCR system using the SYBR Green qRT-PCR
master mix (TaKaRa) and GAPDH as an internal control. The primers are listed in Additional
file 1: Table S1. All Data were processed using the 2
^???CT
method.

MicroRNA mimics, siRNA transient transfection

MicroRNA-106b mimics (sense 5?-UAAAGUGCUGACAGUACAGUGCAGAU-3? and anti-sense 5?-AUUUCACGACUGUCACGACUA-3?),
miR-106b inhibitor (5?-AUUUCACGACUGUCACGACUA-3?), the negative control (5?-CAGUACUUUGUGUAGUACAA-3?),
PTEN-shRNA (sense 5?-GAGCGUGCAGAUAAUGACAdTdA-3? and anti-sense 3?-dAdT CUCGCACGUCUAUUACUGU-5?)
and p21-shRNA (sense 5?-GAAAUAAACGGGACUGAAA dTdT-3? and anti-sense 3?-dTdTCUUUAUUUGCCCUGACUUU-5?)
were purchased from GenePharma (Shanghai, China). The transfection was performed using
Lipofectamineâ„¢ 2000 (Invitrogen, USA) according to the instructions provided by the
manufacturer. Twenty-four or 48 h after transfection, the cells were harvested for
further experiments.

Construction of plasmid vectors and transfection

The coding region of PTEN and p21 was PCR-amplified from human genomic DNA using primer
pairs (PTEN primers forward, 5?-aaggatccCCAGACATGACAGCCATCATC-3? and reverse 5?-cacaactcgagTCAGACTTTTGTAATTTGTGTATGC-3?
and p21 primers forward, 5?-aaggatccGGCGCCATGTCAGAACCGGCTGGGGATGT-3? and reverse 5?-cacaactcgagTTAGGGCTTCCTCTTGGAGAAGATCAG-3?),
digested with BamHI and XhoI and ligated to the pcDNA3.1 vector (Additional file 2). The transfection was performed using Lipofectamineâ„¢2000 (Invitrogen, USA) according
to the instructions provided by the manufacturer.

Viral vectors

The Viral vectors were purchased from GENECHEM company, Shanghai, China (Additional
file 3).

Dual-luciferase reporter assay

The sequences of 3?UTR PTEN and were PCR-amplified from human genomic DNA using primer
pairs (PTEN primers forward, 5?-cacaactcgagTGGCAATAGGACATTGTGTCA-3? and reverse 5?-aaggatccAACAACAAGCAGTGACAGCG-3?
and p21 primers forward, 5?-cacaagtcgacTCCGCCCACAGGAAGCCTGCAGTCC-3? and reverse 5?-aaggatccTTACAAGTAAAGTCACTAAGAATCA-3?),
digested with BamHI and XhoI and ligated to the pLuc vector. They were then cloned
downstream of the Firefly luciferase stop codon in the pLuc control vector (Promega).
All constructs were verified by DNA sequencing (Additional file 4). Cells were seeded in 48-well plates. After 24 h of incubation, the cells were co-transfected
with 1 mg of 3?UTR-PTEN or with 3?UTR mut-PTEN combined with the control oligonucleotide
(final concentration of 80 nM), mimics (80 nM) or inhibitor (80 nM) using Lipofectamine
2000 (Invitrogen) according to the manufacturer’s protocol. Forty-eight hours after
transfection, the Luciferase activity was measured using the Dual Luciferase Reporter
Assay System (Promega). All transfection experiments were conducted in triplicate
and repeated independently 3 times.

MTT assay

The viable cell numbers were measured with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay. The cells were plated in 96-well plates and incubated. Twenty
microlitres of 5 mg/mL MTT (Sigma, USA) was added to each corresponding test well,
and the mixture was incubated for 2 h in a 37°C incubator. The supernatant was then
discarded, and 100 ?l of DMSO (dimethyl sulphoxide) was added to each well to dissolve
the formazan. The optical density (OD) was evaluated by measuring the absorbance at
450 nm of each well, which was read on a spectrophotometer.

Survival foci formation assay

A predetermined number of cells were seeded in 6-well culture plates, and the cells
were then incubated for 24 h to allow settling. The cells were treated with a range
of IR doses [0, 2, 4, 6 and 8 Gy, Nasatron (Cs-137) irradiator]. After incubation
at 37°C for 14 days, the cells were washed twice with PBS and stained with Giemsa
solution. The number of colonies that contained ?50 cells was counted under a microscope
using the following formula: plate clone formation efficiency = (number of colonies/number
of cells inoculated) × 100%. The survival fractions (SF) were calculated by normalizing
the data to the plating efficiency of appropriate control groups. We used GraphPad
Prism (GraphPad Software, LaJolla, CA, USA) to fit the cell survival curve in accordance
with a standard linear-quadratic (LQ) model and obtain the values of the survival
fraction of a range of IR doses.

Flow cytometric analysis of apoptosis

One million cells were harvested and washed twice with cold PBS, fixed in ice-cold
70% ethanol, and incubated overnight at ?20°C. The cells were then stained with 40 ?g/ml
of propidiumiodide (PI) for 30 min. The cells were collected and analysed with the
Cell Quest software (Becton–Dickinson Co, NJ, USA). The percentage of cells with apoptotic
nuclei (% apoptosis) was calculated. Each experiment was performed in triplicate.

Cell-cycle analysis

The cells were harvested, washed twice with cold PBS and fixed overnight at 4°C in
70% ethanol. After the cells were washed twice with PBS, their DNA was stained with
the Cell Cycle Detection Kit (KeyGen, Nanjin, China). The samples were quantified
by flow cytometry (Becton–Dickinson, NJ, USA) and results were analysed with the Modfit
LT software (Verity Software House, Topsham, ME, USA) according to the manufacturer’s
instructions.

Tumour sphere formation assay

The cells were digested with 0.25% trypsin (Sigma, St. Louis, MO, USA), washed twice
with calcium/magnesium-free PBS, suspended in sphere formation medium (DMEM-F12 50 ml + 100 g/ml
EGF + 100 g/ml bFGF + B27 supplement 1 ml), and seeded in 6-cm or 6-well plates (3,000 cells/ml).
The cells were cultivated for 5–7 days (depending on the cell type), and the spheres
were then counted under a microscope.

Immunofluorescence

The cells were seeded on cover slips overnight, fixed with 4% paraformaldehyde for
30 min and treated with 0.25% Triton X-100 for 15 min. After blocking in 10% normal
blocking serum at room temperature for 10 min, the slides were incubated with antibodies
at 4°C overnight followed by washing with PBS three times. The cover slips were then
incubated with Texas Red (TR)-conjugated antibodies for 30 min at room temperature,
followed by staining with 6-diamidino-2-phenylindole (DAPI; Invitrogen).

Protein isolation and western blotting

The cells were washed twice with cold phosphate-buffered saline (PBS) and lysed on
ice in RIPA buffer with 1% PMSF (KeyGen, Nanjin, China). The protein lysates were
resolved on 10% SDS polyacrylamide gels, transferred to PVDF membranes and blocked
in 0.1% Tween20 and 5% skim milk protein in Tris-buffered saline. The membrane was
incubated with primary antibodies (E-cadherin, Vimentin, CD133, Sox2, p21, PTEN, AKT,
p-AKT and ?-H2AX purchased from Epitomics company) followed by incubation with HRP-labelled
rabbit IgG, and the proteins were detected via chemiluminescence. The membrane was
then washed and visualized with horseradish peroxidase (HRP)-conjugated secondary
antibodies for 1 h. The signals were detected with enhanced chemiluminescence (KeyGen,
Nanjin, China).

In vivo tumour growth and xenograft tumour radiosensitivity assay

Athymic nude mice aged 4–6 weeks (GuangDong Experimental Animal Centre) were used
for tumour implantation. All animal experiments strictly adhered to the Regulations
for the Administration of Affairs Concerning Experimental Animals outlined in the
Chinese national guideline for animal experiments issued in 1988. All procedures that
involved animals and their care in this study were approved and performed by the Southern
Medical University Institutional Animal Care and Use Committee (Permit Number: 44007200000784).
The cells were harvested by trypsinisation, washed twice with cold serum-free medium,
and re-suspended with 200 ?l serum-free medium. For the xenograft tumour assay, 2 × 10
⁶
 cells were subcutaneously injected into the backs of nude mice. After tumours were
detected, the tumour size was measured with a slide calliper, and the tumour volume
was determined with the following formula: 1/2 × length × width
²
. Each group consisted of six athymic nude mice, and the mean tumour volume ± SD of
each group was calculated. The harvested tumours were imaged immediately after sacrifice.
To evaluate tumour radioresistance in vivo, the mice were exposed to 8 Gy X-rays when
the tumours reached an average volume of approximately 200 mm
³
. The tumour inhibition rate was recorded and calculated every 4 days: Inhibition
rate = (1 ? irradiation treatment group/control group) × 100%.

Immunohistochemistry

After deparaffinisation and rehydration, the slides were incubated with 3% hydrogen
peroxide solution for 10 min. After a washing procedure with the supplied buffer,
the tissue sections were repaired for 40 min with ethylenediamine tetraacetic acid.
The slides were again incubated with the primary antibody overnight at 4°C. The primary
polyclonal rabbit antibody (Bcl-2 and Bax, Bioword) was diluted at 1:100. The slides
were then incubated with the secondary antibodies (anti-rabbit, DakoCytomation). The
tissue staining was visualized with DAB (DakoCytomation). The slides were counterstained
with haematoxylin and dehydrated. Phosphate buffered solution (PBS) was used as the
primary antibody for the negative controls. Two pathologists each reviewed the results
of immunostaining.

Statistical analysis

Student’s t test and one-way ANOVA analysis were performed to assess statistical significance.
Spearman’s correlation coefficient was calculated to test the association between
miR-106b and PTEN in the classes normal versus tumour. The results of all experiments
are expressed as the mean ± standard deviation SD of 3 independent experiments. P
values0.05 were considered statistically significant.