MiRNA-149 modulates chemosensitivity of ovarian cancer A2780 cells to paclitaxel by targeting MyD88

Cell line and maintenance

The A2780 cell line was obtained from the Institute of Cell Biology (Shanghai, China).
The cells were maintained in RPMI 1640 medium (Gibco-BRL, Carlsbad, CA, USA) supplemented
with 10 % fetal bovine serum (FBS), 100 U/ml penicillin and 100 U/ml streptomycin
at 37 °C in a humidified incubator with 5 % CO
₂
. The cells were demonstrated to be free of mycoplasma.

Construction of miRNA-149 inhibitor and MyD88 lentiviral vectors

MiRNA-149 inhibitor (5’-GGGAGUGAAGACACGGAGCCAGA-3’) was inserted into the LV3-pGLV-H1-GFP/puro
lentiviral vector, and siRNA (5’-UUCUCCGAACGUGUCACGUdTdT-3’) was used as a negative
control. MyD88 whole cDNA synthesized by GenePharma (GenePharma, Shanghai, China)
was subcloned into the LV5-pGLV-EF1a-GFP/Puro Lentiviral plasmid vector. Lentiviruses
expressing inhibitor against miRNA-149, MyD88, and the controls were produced by co-transfection
of 293 T cells using polybrene (GenePharma, Shanghai, China) according to standard
protocols. A2780 (5 × 10
⁴
) cells were infected with lentivirus at a MOI (multiplicity of infection, pfu number/cell)
of approximately 100 for 24 h. Cells were then transferred into complete medium.

RNA isolation and reverse transcription polymerase chain reaction (RT-PCR)

Small RNAs were purified from differently treated A2780 cells using an RNA purification
kit (TIANGEN Biotech, Beijing, China). Total RNA was extracted with Trizol reagent
according to the protocol described by the supplier (TakaRa, Dalian, China). First-strand
cDNA was synthesized from 1 ?g of total RNA in a 20-?l reaction mixture using the
PrimeScript RT reagent kit (TakaRa, Dalian, China). Quantitative real-time PCR-based
gene expression analysis was performed on a real-time PCR instrument (7300, Step One
Plus, Applied Biosystems, USA) using a standard SYBR-Green PCR kit. The parameters
used for all PCR reactions were as follows: One cycle of 95 °C for 2 min, followed
by 40 cycles of 95 °C for 15 s, and 60 °C for 30 s. Specific primer sets were used
for RT-PCR of the U6 control, miR-149, ?-actin control, and MyD88. The relative expression
of each target gene was calculated using the 2
^-??ct
method.

Analysis of apoptosis

The percentage of apoptotic cells was quantitated using the Annexin V-PE Kit (Becton-Dickinson)
according to the manufacturer’s instructions. Stained cells were analyzed by flow
cytometry during the first 30 min of staining. ?10,000 cells were measured using a
FACScan instrument (Becton-Dickinson) and the data were analyzed using FlowJo software
(Tree Star Inc.).

Cytotoxicity assay

Cell viability was assessed using the Cell Counting Kit 8 (CCK-8) (Dojindo Laboratories,
Kumamoto, Japan) assay. 6 × 10
⁴
A2780 cells (100 ?l) were seeded into 96-well plates. After 24 h of incubation, paclitaxel
(Sigma) was added at concentrations of 0 ?M, 0.1 ?M, 0.2 ?M, 0.3 ?M, 0.4 ?M and 0.5 ?M
(each concertration were done repeat five experiments); the cells were then incubated
for 48 h. After 48 h, 10 ?l of CCK-8 solution was added to each well, followed by
4 h of incubation at 37 °C. The OD values were read at dual wave lengths of 450 nm
and 630 nm to determine cell viability using a microplate reader (Thermo Fisher Labsystems).

Analysis of cell cycle

Cells (2?×?10
⁶
) were pelleted by spinning for 5 min at 1000 rpm and 4 °C and resuspended in 1 ml
of cold PBS. After fixation by adding 4 ml of absolute ethanol, the cells were centrifuged
and resuspended in 1 ml of PBS. Then, 100 ?l of 200 ?g/ml DNase-free RNaseA was added
to the cell suspension and incubated for 30 min at 37 °C. The cells were stained with
100 ?l of 1 mg/ml propidium iodide (light sensitive) and incubated for 5–10 min at
room temperature before analysis.

Western blot

The protein concentration of each sample was determined by BCA Protein Assay Kit (Beyotime,
Jiangsu, China). Equal amounts of protein were loaded and separated discontinuously
on 12 % sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), and subsequently transferred
onto a PVDF membrane (Amersham Pharmacia, UK). The membrane was then incubated in
TBST blocking solution (Tris-buffered saline including 0.1 % Tween-20) containing
5 % skim milk for 2 h at room temperature, followed by separate incubation with primary
antibodies against Bax, MyD88, Bcl-2 (Cell Signaling, USA) and ?-actin (Beyotime,
Jiangsu, China) overnight at 4 °C. After washing, the membrane was incubated with
HRP-conjugated anti-mouse, anti-rabbit, or anti-goat secondary antibodies for 2 h.
After several washes, the immunoblot was detected with enhanced chemi-luminescence
reagent (Pierce Biotechnology, USA) according to the manufacturer’s instructions.

Immunocytochemistry

Expression of MyD88 protein was determined using immunocytochemistry. In total, 1?×?10
⁵
cells were seeded into a Millicell EZ Slide (Millipore, Shanghai, China). After 24 h
of incubation, cells were fixed on slides using 4 % paraformaldehyde. The cells were
permeabilized for 10 min with 0.1 % Triton X-100 in PBS and blocked with blocking
buffer (2 % BSA, 0.1 % Triton X-100) for 30 min at room temperature. After blocking,
the cells were washed with PBS and incubated overnight with rabbit anti-human MyD88
antibodies (Sigma, Shanghai, China) at 4 °C. On the following day, the cells were
washed three times with PBS, and then incubated with Cy3-labeled goat anti-rabbit
IgG (H?+?L) (Beyotime, China) for 1 h followed by washing with PBS three times.

Cell migration assay

Cell migration were evaluated using the Transwell Permeable Support (Corning) according
to the manufacturer’s instructions. Five 200-multiple microscopic fields were randomly
selected to calculate the total count of the invaded or migrated cells. All assays
were conducted three times.

Statistical analysis

For all analyses, the measurements obtained from the groups were expressed as the
means?±?SD for all data determined. Statistical analysis was performed using an unpaired
Student’s t-test followed by Tukey’s test. P??0.05 was considered statistically significant.