MIR-147B REGULATED PROLIFERATION
AND APOPTOSIS OF GASTRIC CANCER CELLS
BY TARGETING CPEB2 VIA THE PTEN PATHWAY
Tao K.1,2, Dong J-H.2, Wang D.1, Li F.2†#, Zhang Z-T.#1*
*Corresponding Author: Zhong-Tao Zhang, MD, Email: sxzhangzhongtao@sina.com, ORCID ID: 0000-0002-1184-2591
#: Zhong-Tao Zhang and Feng Li contributed equally to the article
†: Co-corresponding author: Feng Li, Email: sxlifengwobuxin@sina.com, ORCID: 0000-0002-7322-422X
page: 10
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MATERIAL AND METHODS
Materials
The five Gastric Cancer cell lines, i.e., SGC-7901,
MGC-803, BGC-823, AGS and MKN-45 and normal gastric
tissue cell lines were obtained from the sample bank
at Shanxi Tumor Hospital. The specific information of 50
pairs of gastric cancer specimens in the biological sample
bank is related to gastric body cancer patients who underwent
radical gastrectomy at the Shanxi Tumor hospital
from January 2014 to January 2015. Of these patients,
29 were male and 21 female. The age ranged from 43 to
70 years. The TNM system was used for clinical staging.
There were 20 cases of stage I+II and 30 cases of stage III.
Inclusion criteria:
1. Gastric body cancer
2. Total gastrectomy
3. 3.ECoG score ≤ 1 point.
Exclusion criteria
1. >70 years old
2. Patients with more serious complications or chronic
diseases requiring consultation and treatment
3. Other tumor history or related treatment history
4. Serious complications which occurred after operation.
5. Follow up data were missing.
The tissue samples taken in this study were obtained
from the patients after their written consent was obtained.
This study has been duly approved by the medical ethics
committee of the Beijing Friendship Hospital, Capital
Medical University.
Methods
The GC tissues of 50 patients with gastric cancer with
complete data and the adjacent tissues > 5cm were divided
into an experimental group and a control group in order to
determine the expression level of the target gene miR-147b
in the selected tissues. BGC-823, SGC-7901, AGS, MGC-
803 and MKN-45 GC cell lines served as markers for the
experimental group, whereas normal gastric tissue cell
lines serve as the control group to quantify the expression
of miR-147b. Two groups of cell lines with high expression
of miR-147b were selected for transfection experiment: 20
~ 50 nM miR-147b inhibitor was used as the experimental
group and miR-147b NCas was used as the negative control
group. We measured the cell cycle and colony formation
after 24h of transfection and cell migration after 48h of
transfection of the two groups of transfected cell lines. The
proliferation rate of GC cells at 0, 24, 48, 60, and 72 hours
after transfection was determined by the CCK8 method.
Cell resuscitation and culture
All cell lines were maintained in complete Dulbecco’s
Modified Eagle Medium, supplemented with 10% (vol./
vol.) fetal calf serum, penicillin G (100 μg/mL) and streptomycin
(100 μg/mL). All cell lines were grown at 37°C in
humidified 5% CO2 and 95% air atmosphere. All cell lines
were incubated for 24h prior to experiments.
RNA isolation and quantitative qrtPCR
Three pairs of gastric cancer tissues were randomly
selected for tissue microarray to detect the expression of
the target and related genes in gastric cancer tissues. The
expression of the target gene in 50 pairs of gastric cancer
tissues was also determined. Total RNA was extracted from
GC cell lines, normal tissues, and tissues using Trizol reagent
(Invitrogen, USA) to determine the expression of
miR-147b gene. The expression of miR-147b in the cells
of each group was analyzed and compared. miR-147b was
amplified by the upstream primer 5’-GGGGTGTGTGGAAAT-
3’ and reverse primer 5’- AACTGGTGTCGTGGAGTCGGC-
3’, and reverse transcribed into cDNA using
RNA to cDNA premix. QRT-PCR was performed with
SYBR Green master mix according to the protocol provided
(all from vii7 Q-PCR system). The concentration of RNA
was quantified by Nanodrop-2000 (Nanodrop, USA). The
relative expression of miR-147b was calculated by 2-ΔΔCt10.
Transfection experiment
After the PCR quantitative analysis, two groups of
cell lines with high expression of miR-147b were selected
for the transfection experiment. 20 ~ 50 nM miR-147b inhibitor
was used as the experimental group and miR-147b
NC serves as the control group. The cells were cultured
at ambient temperature and humidity at 5 % CO2 for 36
~ 48h. The clone with the highest expression of foreign
protein in monoclonal cells was used in Western blot, and
scratch test, apoptosis test, clone formation test and cell
proliferation were detected.
Scratch test
Exponentially, cells that were growing were trypsinized
and seeded at a density of 200,000 cells per well into
12-well plate for a 24h incubation (~90% confluence). The
scratch wounds were made by a sterile 1 mL pipette tip
through a pre-marked line. After removal of the resulting
debris from the five lineal scratches, the cell monolayer
was subsequently rinsed three times with PBS, followed
by incubation with sample for 24h. The wound areas were displayed by taking images just above the interchanges
between scratched wound areas and pre-marked lines. The
effect of the sample on the wound closure was determined
microscopically (Olympus IX - 7) after 0, 24, and 48h,of
incubation. The effect of the sample on the wound closure
was captured using MRI imaging software.
Apoptosis test
The cells were seeded in 12-well plates (2×105 cells/
well). The cells were cultured and incubated (with 5%
CO2 and 95% air) at 37°C. Various concentrations of test
drugs were dissolved in DMSO and incubated with the
cells for 48h. DMSO in the culture medium never exceeded
0.1% (v/v), the concentration known not to affect
cell proliferation. The Annexin V-FITC/PI apoptosis kit
(Keygen Biotech, China) was used. For this purpose, the
cells were incubated with 5 μl Annexin V-FITC and 5 μl
PI for 5 min in the dark. Data were collected with moflow
(Beckman Coulter, USA).
Clone formation assay
One thousand cells were plated in a 35 mm Petri dish
after a different treatment was applied for 24h and allowed
to grow for two weeks. The medium was changed twice
per week. Then, the colonies were fixed with 4% paraformaldehyde,
stained with methyl violet. The colonies
were observed under a microscope. Colony inhibition rate
= [(1-number of colonies in experimental group / control
group) ×100%; colony formation efficiency = 1-colony
inhibition rate.
Cell proliferation test
Cell suspensions of the two cell lines were inoculated
into 96 well plates, incubated at ambient temperature
and humidity at 5 % CO2, and further cultured after
adding 10μL CCK-8 reagent (Dojindo, Japanese) to
each well. CCK-8, being nonradioactive, allows sensitive
colorimetric assays for the determination of the number
of viable cells in cell proliferation and cytotoxicity assays.
It works on the principle of highly water-soluble
tetrazolium salt. WST-8 [2-(2-methoxy-4-nitrophenyl)-
3-(4-nitrophenyl)-5-(2,4- disulfophenyl)-2H-tetrazolium,
monosodium salt] produces a water-soluble formazan dye
upon reduction in the presence of an electron mediator.
WST8 is reduced by dehydrogenases in cells to give an
orange colored product (formazan), which is soluble in
the tissue culture medium. The amount of the formazan
dye generated by dehydrogenases in cells is directly proportional
to the number of living cells. The absorbance
values of each well were measured at 450 nm and 630
nm at 0, 24, 48, 60 and 72 hours after transfection. The
experiments were conducted three times. Cell viability has
been calculated using the formula * (%) = [a (dosing) - A
(blank)] / [A (0 dosing) - A (blank)] × 100.
Vector construction and luciferase analysis
The target scan database was selected to search for
the target gene that can be combined with miR-147b and
map the base pairs that cross the 3 ‘untranslated region of
miR-147b and the predicted target gene. CPEB2 plasmid
was constructed by the pGL3 promoter vector: wtCPEB2
(5′-GUUUCUGUAUAGAAUCGCACAAG-3′) and mutant
mutCPEB2 (5′-GUUUCUGUAUAGAAUTTCTCGAG3′).
MGC-803 cells were cultured until the cell density
grew to 80% - 90%. The cells were divided into an
experimental group and a control group. Lipofectamine™
2000 transfection reagent was used for co-transfection
of the MGC-803 cells with the wt-CPEB2 plasmid and
scramble or miR-147b inhibitor. The second group was
divided into an experimental group and a control group.
Lipofectamine™ 2000 transfection reagent was used for
co-transfection of MGC-803 cells with mut-CPEB2 plasmid
and scramble or miR-147b inhibitor were co transfected
into MGC-803 cells by using. Double glo stop and
glo reagent were added into 24 well plate cells at 48h.
After full reaction at room temperature, the fluorescence
intensity was detected according to the instructions of
double luciferase reporting system (Promega, USA) and
the luciferase activity in MGC-803 cells was analyzed.
Western blotting
MGC-803 cells were co-transfected with miR-147b
NC, miR-147binhibitor and miR-147b NC+CPEB2 and
were further categorized into two groups to identify the
expression of CPEB2 and GAPDH (control group).MGC-
803 and BGC-823 cells were separated into two groups
after the culture. One group was added with an anti-CPEB2
antibody (Abcam, ab222070) to establish the si-CPEB2
and si-control experimental group and control group. After
loading (25 μg/lane), the protein concentration was
estimated using the BCA Kit and protein was separated
using SDS-PAGE. Thereafter, the western transfer was
performed with PVDF membranes (Millipore, Bedford,
MA, USA) using the primary and secondary antibodies
as per the earlier reported procedure. 9
H&E staining and immunohistochemistry
Routine H&E staining and immunohistochemical
analysis was conducted to determine the expression of
CPEB2 in the gastric cancer tissues and was observed
using a Zeiss UV LSM confocal microscope.
Statistical analysis
Statistical analysis was evaluated using a two tailed
t-test and one-way ANOVA with SPSS packaging program (SPSS17.0, USA). The Pearsonχ2 test was used to compare
the differences between groups. If the conditions were not
met, continuity correction χ2 analysis was further used.
Statistical significance was set as P < 0.05.
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