JARID1B Deletion Induced Apoptosis In Jeko-1 And HL-60 Cell Lines

Copy and paste this link to your website, so they can see this document directly without any plugins.



Keywords

JARID1B, with, cell, were, histone, siRNA, Jeko-1, HL-60, cells, H3K4, that, apoptosis, Ki67, expression, cyclin, using, proliferation, nmol/L, methylation, Pathol, nmol/L,, from, modulated, hours, which, Clin, 2015;8(1):171-183, lines, folds,, Cell

Transcript

Int J Clin Exp Pathol 2015;8(1):171-183
www.ijcep.com /ISSN:1936-2625/IJCEP0002873
Original Article
JARID1B deletion induced apoptosis in Jeko-1 and
HL-60 cell lines
Haiyan Su1, Xudong Ma2, Yiqun Huang2, Huidan Han2, Yong Zou2, Wenhua Huang3
1Department of Pathology, Zhangzhou Affiliated Hospital of Fujian Medical University, Zhangzhou, Fujian, China;
2Department of Hematology Zhangzhou Affiliated Hospital of Fujian Medical University, Zhangzhou, Fujian, China;
3School of Basic Medical Sciences, Southern Medical University, Guangzhou, China
Received September 29, 2014; Accepted December 20, 2014; Epub January 1, 2015; Published January 15,
2015
Abstract: Aims: To investigate the involvement of JARID1B histone methyltransferase in the epigenetic change of
euchromatic promoter in mantle cell lymphoma (MCL) and acute leukemia. Methods: We retrospectively analyzed
the protein of JARID1B and tri-methylated histone H3 lysine 4 (H3K4), histone H3 lysine 9 (H3K9), and cyclin D1
and Ki67 in 30 cases of MCL by immunohistochemistry. JARID1B was depleted by small interfering RNA (siRNA),
and cell apoptosis and cell proliferation were detected by flow cytometry and MTT [3-(4,5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide], histone tri-methylated H3K4 and histone acetylated H3, H4, cyclin D1, Bcl-2, procaspase-3, C-myc were studied by Western blot. Results: We demonstrated that JARID1B was upregulated and
histone tri-methylated H3K4 was downregulated in MCL compared to proliferative lymphadenitis, P < 0.05. The
expression of histone methylated H3K9 was similar in both. Histone methylation of H3K4 was positively correlated
with Ki67 in MCL (Kappa = 0.757, P < 0.05). This study showed that depletion of JARID1B cleavage apoptotic proteins of Bcl-2, procaspase-3, C-myc and resulted in loss cell viability and inducing apoptosis in Jeko-1 and HL-60
cell lines. JARID1B siRNA improved tri-methyl H3K4 and histone acetylated H3 and inhibited cyclin D1, but did not
affect histone acetylated H4. Conclusions: This study revealed hyper JARID1B expression and hypo histone H3K4
tri-methylation in MCL. We identify depletion JARID1B as a demethylase which is capable of removing three methyl
groups from H3K4 and up-regulating histone acetylation of H3 in both cell lines. Interestingly, depletion of JARID1B
inhibits Cyclin D1, which is one of the genes contributes to MCL pathogenesis. JARID1B might be one of therapeutic
targets in acute leukemia and MCL.
Keywords: siRNA, JARID1B, histone methylation, MCL, acute leukemia
Introduction
Epigenetic markers, like DNA methylation and
histone modifications, contribute to physiological and pathological states, including cancer
[1]. Aberrant histone methylation has not been
well characterized in human disease, especially
in MCL. JARID1B, also named Plu-1, is one of
the four members of the JARID1 protein family,
which catalyzes the removal of three methyl
groups from lysine 4 of histone H3 [2, 3]. A
recent discovery shows that H3K4 trimethylation is associated with active gene repression
and suggests that H3K4 methylation may have
a spatial and temporal effect on transcription.
JARID1B has been shown to be up-regulated in
breast cancer and prostate cancer, and probably involved in their development and progression [4, 5]. JARID1B associates with the oncogene ZNF217 as part of a larger complex which
also includes LSD1, and represses gene transcription [6] and is involved in cell cycle and signal transduction [7].
MCL is a well-defined lymphoid neoplasm characterized by a proliferation of mature B lymphocytes carrying the t(11;14)(q13;q32) translocation that leads to the overexpression of cyclin
D1 [8]. It is an incurable, combining the unfavorable clinical feature of aggressive and indolent
lymphomas. Conventional therapy can only
make 10%-15% of patients in complete remission. Acute myeloid leukemia is the most common acute leukemia in adults. Chemotherapy
induces a complete remission in 70%-80% of
younger patients (age, 16 to 60 years), but
JARID1B modulated apoptosis in Jeko-1 and HL-60 cell lines
172 Int J Clin Exp Pathol 2015;8(1):171-183
many of them have a relapse and die of their
disease. There is some acute leukemia with
cytogenetic translocations in WHO classification, which involved in epigenetic modification
change.
In this report, we investigated the state of
JARID1B and histone methylation of H3K4 and
H3K9 in MCL, evaluated the correlation with
histone methylation of H3K4 and Ki67 that is
maker of prognosis in MCL. Further we depleted JARID1B by siRNA technique and measured
consequent histone modification, gene transcription, cell proliferation and apoptosis in
Jeko-1 and HL-60 cell lines.
Materials and methods
Collection of patients’ samples and data
This study consisted of a total of 30 cases of
MCL patients who hospitalized at the
Zhangzhou Affiliated Hospital of Fujian Medical
University between January 2005 and
December 2010. The lymphoid tissue samples
were collected from all participants after
obtaining written informed consent, which was
approved by the Institutional Review Board at
the hospital. Diagnosis is made according to
the WHO classification. A total of 30 cases in
MCL was overexpression of cyclin D1. Patients’
samples with hyperplastic lymphadenitis (n =
30) were used as control.
Materials and reagents
Antibodies for rabbit against human tri-methylhistone H3K9, rabbit against human tri-methylhistone H3K4, rabbit against human JARID1B
were purchased from Upsate Biotechnology
(Lake Placid, NY, USA); Antibodies for cyclin D1
came from Dako (EP12), Ki67 from Dako
(MIB1).
Tissue microarray (TMA) construction and immunohistochemistry
A representative tumor paraffin block (donor
block) was collected from each case, and two
tissue cores (2 mm in diameter) were obtained
using a trephine apparatus. Trephinated paraffin tissue cores were then arranged in a new
recipient paraffin block (tissue array block).
Cores containing tumor in more than 50% of
the area were considered adequate. Immunohistochemical staining was performed using
commercially polyclonal rabbit antibodies to
histone methyltransferase JARID1B, tri-methylhistone H3K9 and H3K4, Ki67, cyclin D1.
Tissue array blocks were section at a thickness
of 4 μm and mounted on precoated glass
slides. The sections were deparaffinized and
hydrated prior to immunohistochemistry. The
immunohistochemical S-P method was performed according to the manufacturer’s protocol. Tissues positive for all the purchased antibodies were used as positive controls, sections
prepared with phosphate buffer saline instead
of the primary antibody were used as negative
controls. Positive control exhibited brown color
in the nuclei. Chevallier’s semi-quantity system
analysis was used in the calculation of immunohistochemistry results. For JARID1B, tri-methylhistone H3K9 and H3K4, the results are presented as the sum of scores presenting color
density and the percentage of stained cells.
According to color density, non-stained cells
were scored as 0, slightly stained were 1, intermediate-stained were 2, strongly stained were
3. When the number of positive cells was <
25%, 25%-50%, or 50%-75%, or > 75%, the
immunoreactivity was scored as 1+, 2+, 3+, 4+
respectively. The two scores of presenting color
density and the number of positive cells were
added for each slide. The sum that was 0 was
negative, 1-2 was presented as +, 3-4 as ++,
5-6 as +++, and 7 as ++++. If the sum of the
two scores was less than 2, it was considered
negative staining; more than two was considered positive staining. The scores of each
patient group were averaged.
The Ki67 index was defined as the percentage
of Ki67-positive tumor cells in representative
areas of the lymphoma. To count the number of
Ki67-positive cells, two representative areas
were chosen. A representative area was defined
not to contain residual germinal centers, hot
spots of proliferation or proliferating T cells. Hot
spots of proliferation were areas of tumor cells
of less than two high-power fields in size (field
of vision at × 400 magnification), which proliferate higher than the rest of the tumor. Counting
was performed by one observer. The positive
cells among 500 cells were counted using an
eyepiece with a grid in a × 40 objectives. The
Ki67 index was calculated as the percentage of
positive cells by averaging the values obtained
for the two areas (count-Ki67 index) with a
JARID1B modulated apoptosis in Jeko-1 and HL-60 cell lines
173 Int J Clin Exp Pathol 2015;8(1):171-183
Figure 1. Expression of histone H3K4, H3K9 methylation, JARID1B, Ki67 and cyclin D1 in MCL. Histone H3K9, H3K4 methylation, JARID1B, Ki67 and cyclin D1
were detected by Immunohistochemistry. Tissue microarray was constructed and immunohistochemical staining was performed. Chevallier’s semi-quantity system
analysis was used in the calculation of immunohistochemistry results in histone H3K4, H3K9 methylation, JARID1B and cyclin D1. Ki-67 values obtained via counting of 500 cells using an eyepiece with a grid in a × 40 objectives.
JARID1B modulated apoptosis in Jeko-1 and HL-60 cell lines
174 Int J Clin Exp Pathol 2015;8(1):171-183
Table 1. Expression of H3K4, H3K9 and JARID1B in MCL
MCL Control
Number - + ++ +++ rate - + ++ +++ rate X2 P
H3K4 30 0 11 12 7 23% 0 4 9 17 57% 6.94 < 0.01
H3K9 30 0 3 1 26 87% 0 0 1 29 97% 0.87 > 0.05
JARID1B 30 0 11 8 11 37% 0 12 14 4 13% 4.35 < 0.05
Chi-square was done to compare the positive rate of H3K, H3K9, and JARID1B in MCL and
control.
semiquantitative evaluation system, consisting
of four levels: < 10%, 10-30%, 30-50% and >
50% labeled cells. Negative control staining
was obtained by omitting the primary
antibodies.
Cell culture
Human MCL Jeko-1 and HL-60 cell line were
purchased from Shanghai Institute of Cell
Bank. Cells were cultured in 10% fetal bovine
serum, and RPMI1640 containing 2 mM
L-glutamine under 37°C, saturated humidity
and 5% CO2. Cells were subcultured every 3-5
days. Cells were seeded and treated with the
desired siRNA concentrations in different time
points.
RNA interference
The approach by transient transfecting using
LipofectamineTM 2000 was employed to deplete
JARID1B expression from Jeko-1 and HL-60 cell
lines. JARID1B siRNA sense: 5’-GGAGCUAUUCAAUUAACUATT-3’ Anti-sense 5’-UAGUTotal RNA was extracted from samples of 1 ×
106 cells using TRIzol reagent (Invitrogen). The
quantity and quality of RNA samples were measured by absorbance at 260 and 280 nm. RNA
samples with an A260:A280 ratio 1.8~2.0
were stored at -80°C until use. Copy DNA synthesis was performed using an Avian
Myeloblastosis Virus Reverse Transcriptase kit,
according to manufacturer’s protocol (Promega,
Madison, WI, USA). PCR β-actin was used as
internal control. Primers used in PCR amplifications were: JARID1B forward, 5’-ATAGAATTCGGGAATCTTAAATTTG-3’; JARID1B reverse, 5’TATCTCGAGTTCCTGTTCGGAATAGG-3’; β-actin
forward: 5’-ATGTCACGCACGATTTCCCGC-3; βactin reverse: 5’-GGCATGGGTCAGAAGGATTCC3’. Amplicon size was 580 base pairs (bp) and
420 bp for JARID1B and β-actin respectively.
PCR reaction conditions were: 95°C 30 seconds, 60°C 30 seconds, 72°C 40 seconds, and
was repeated 32 cycles. PCR-amplified products were electrophoresed in 1.5% (w/v) agarose gels with 1 μg/ml ethidium bromide.
Experiments were repeated twice.
Table 2. Expression of Ki67 in MCL
Ki67
< 10% 11-30% 31-50% > 51% mean ± SD P
MCL 20 (66.7%) 5 (16.7%) 2 (6.7%) 3 (10%) 12.53 ± 23.70 < 0.05
Control 28 (93.3%) 2 (6.7%) 0 0 1.97 ± 2.70%.
WILCOXOM rank sum test was done to compare the positive degree of ki67 in MCL and control (Z = -2.65 P < 0.05).
Table 3. Positive staining of H3K4 and Ki67 in MCL
Ki67
+ -
H3K4 + 7 0
- 3 20
Ki67 > 10% and H3K4 +++ are given as positive. (Kappa = 0.757, P < 0.05)
Cohen’s kappa was measured agreement between the positive staining of H3K4 and Ki67
in MCL.
UA AUUGA AUAGCUC -
CAG-3’ were synthsized
by Shanghai GenePharma Co. Ltd. (China).
Transfection with siRNA
was performed according to the LipofectamineTM 2000 manufacturer’s protcol (Invitrogen, Carlsbad, CA). Inducible Jeko-1 and HL-60
cells (1 × 105 cells/well)
were seeded onto 24well plates, (Costar Life
Science, Acton, MA) and
transiently transfected
with desired concentration of JARID1B siRNA.
All experiments were
conducted in triplicate using independent cultures. Both total RNA
and protein were extracted after 24 hours
transfection.
Reverse transcription
polymerase chain reaction (PCR)
JARID1B modulated apoptosis in Jeko-1 and HL-60 cell lines
175 Int J Clin Exp Pathol 2015;8(1):171-183
Evaluation of apoptosis by flow cytometry
To detect the phosphatidylserine translocation
on the surface of plasma membrane, cells were
seeded in 6-well plates (1 × 106 cells/well,
Costar Life Science) with sterile cover glass
placed at the bottom of each well, and then
transfected with negative control siRNA, 60,
120, 240 nmol/L JARID1B siRNA for 24 hours
while cells at logarithmic phase. Cells were collected, resuspended in Annexin V binding buffer and then incubated with Annexin V-FITC and
PI following the manufacture’s instruction(BD)
by flow cytometry (Epics-XL, Beckman). 10000
events per sample were acquired on the FAC
Scan. Fluorescent commissions were collected
Cell proliferation measured by MTT
The Jeko-1 and HL-60 cells were seeded at a
density of 1 × 105/well in 96-well culture dishes
(Costar Life Science). After 0, 24, 48, 72 and
96 hours transfection (n = 5) with indicated
concentration of JARID1B siRNA, cell proliferation was measured using MTT assays (0.5 mg/
ml; Sigma). The spectrophotometric absorbance of the samples was determined by using
Ultra Multifunctional Microplate Reader (Tecan,
Durham, NC) at 492 nm and 630 nm for absorbance (OD value). Suppression ratio was also
calculated. The experiment was repeated in
triplicated. Cell proliferation rate (%) = (A experiment-A blank)/(A control-A blank) × 100%.
Figure 2. A. Jeko-1 cells and HL-60 cells were transfected with JARID1BsiRNA. A. Jeko-1 cells were transfected with
JARID1B siRNA after 24 hours in fluorescence microscope (left), transfection efficiency was 96% ± 3.31% (n = 5).
HL-60 cells were transfected with JARID1B siRNA after 24 hours in fluorescence microscope (right), transfection efficiency was 93% ± 2.56% (n = 5). B. The expression of JARID1B mRNA by different concentrations of siRNA in Jeko1 and HL-60 cell line. After 24 hours transfection of JARID1B, the amplification of JARID1B mRNA attenuated with
concentration dependent manner. Amplification of JARID1B was (1.07 ± 0.15) with 60 nmol/L, (0.63 ± 0.17) with
120 nmol/L, (0.22 ± 0.7) with 240 nmol/L respectively in Jeko-1, compared to control (1.25 ± 0.13), χ² = 12.81,
P < 0.05 (upper). Amplification of JARID1B was (0.87 ± 0.15) with 30 nmol/L, (0.56 ± 0.09) with 60 nmol/L, (0.12
± 0.03) with 120 nmol/L respectively in HL-60, compared to control (1.13 ± 0.15), χ² = 23.25, P < 0.05 (lower).
JARID1B modulated apoptosis in Jeko-1 and HL-60 cell lines
176 Int J Clin Exp Pathol 2015;8(1):171-183
standard deviation. The t test was done to compare the levels of the parameters between the
two groups. Different between the valus were
assessed for statistical significance by Chisquare, WILCOXOM rank sum test, one-way
ANOVA and repeated measure ANOVA.
Agreement measure were calculated by
Cohen’s kappa. P < 0.05 was considered statistically significant.
Results
Overexpression of JARID1B and lower expression of histone tri-methylation of H3K4 in MCL
We assessed the staining score of JARID1B,
histone methylated H3K4 and H3K9 for 30
cases of MCL. The stained position was in cell
nucleus. The expression of JARID1B in MCL
was 37% (11/30), higher than that in hyperplastic lymphadenitis of 13% (4/30), P < 0.05.
The expression of histone tri-methylation of
H3K4 in MCL was 23% (7/30), lower than that
in hyperplastic lymphadenitis of 57% (17/30), P
< 0.01. The expression of histone tri-methylation of H3K9 in MCL was 87% (26/30), with
similar to hyperplastic lymphadenitis of 97%
(29/30), P > 0.05 (Figure 1, Table 1). The
expression of cyclin D1 in MCL was 100% positive, but that in hyperplastic lymphadenitis was
100% negative (Figure 1).
Correlation of histone methylated H3K4 with
Ki67 in MCL
Ki67 labeling index was expressed 100% in
MCL, including with ≤ 10% in 66.7% (20/30) in
Figure 3. JARID1B siRNA induce cell proliferation in Jeko-1 and HL-60 cells. 1 × 105 cells were seeded into a 96-well
culture dishes and treated with JARID1B siRNA in different concentration and time point. Cell proliferation was measured using MTT assays (0.5 mg/ml; Sigma). The spectrophotometric absorbance of the samples was determined
by using Ultra Multifunctional Microplate Reader (Tecan, Durham, NC) at 492 nm and 630 nm for absorbance (OD
value). Suppression ratio was also calculated. The experiment was repeated in triplicated. A. Cell proliferation was
conducted in different concentrations in Jeko-1 and HL-60 cells.Repeated measure ANOVA: F = 109.3 P < 0.01 B.
60 nmol/L JARID1B siRNA treated to the cells in different time in Jeko-1 and HL-60 cells.Repeated measure ANOVA:
F = 115.1 P < 0.01.
through 5.30 and 5.70 nm had pass filters for
FITC and PI respectively.
Western blot analysis
Total protein lysates and Western blotting analysis were performed as previously described
[9]. Briefly, Jeko-1 and HL-60 cells were plated
on culture dishes and transfected by JARID1B
siRNA with 60, 120, 240 nmol/L for 24 hours.
Control cells were incubated in the medium
with Na2CO3 using same time points. After incubation, total proteins were prepared from each
culture condition with a lysis buffer containing
freshly prepared protease inhibitors. Protein
concentration was then measured using BCA
protein assay (Pierce, Rockford, IL). Cell extracts
were subjected to 12% SDS-PAGE and electrophoretically transferred to nitrocellulose membrane. Membranes were incubated with monoclonal anti-tri-methylated-histoneH3K4, antihistone acetylation of H3, H4, JARID1B (Upstate,
UAS), BCL-2, proaspase-3, C-myc, cyclin D1
(Santa Cruz USA). After being washed with TBS,
membrane incubated with secondary antibody
conjugated with peroxidase. The signal was
then detected using the chemiluminescent
detection system (Pierce) and analyzed by a
color image analysis system (AlphaDigiDoc),
(Alpha Innotech).
Statistical analysis
All statistical calculations were performed by
SPSS version 18.0 for Windows software (SPSS
Inc, IL, USA). Results are presented as means ±
JARID1B modulated apoptosis in Jeko-1 and HL-60 cell lines
177 Int J Clin Exp Pathol 2015;8(1):171-183
Figure 4. JARID1B siRNA produce cell apoptosis in Jeko-1 and HL-60 cells. 1 × 105 cells were seeded into a 96-well culture dishes and treated with JARID1B siRNA
in 0, 30, 60, 120 nmol/L for 24 hours. Annexin V-PI staining was performed to determine apoptotic cells population. A. Apoptosis was increased gradually after
transfected with siRNA JARID1B in 30, 60, 120 nmol/L for 24 hours respectively in Jeko-1, One-way ANOVA: F = 67.5 P < 0.05. B. Apoptosis was increased gradually
after transfected with siRNA JARID1B in 30, 60, 120 nmol/L for 24 hours respectively in HL-60, One-way ANOVA: F = 34.8 P < 0.05. Fluorescence signals from Annenxin V-FITC and from PI are reported on the x-axis and y-axis, respectively. Numbers shown in the four quadrants represent the percentage of viable (lower left),
necrotic (upper right), early apoptotic (lower right) and late apoptotic (upper right) cells.
JARID1B modulated apoptosis in Jeko-1 and HL-60 cell lines
178 Int J Clin Exp Pathol 2015;8(1):171-183
MCL, 11% to 30% in 16.7% (5/30), 31% to 50%
in 6.7% (2/30), > 50% in 10.0% (3/30). The
mean index was 12.53 ± 23.70%. Ki67 in
hyperplastic lymphadenitis with ≤ 10% was in
93.3% (28/30). Only 6.7% was expressed in
11% to 30%. The mean index was 1.97 ± 2.70%,
P < 0.05 (Figure 1, Table 2). We further analyzed the correlation between histone methylation of H3K4 and Ki67 in MCL. The data showed
that the Ki67 labeling index was positive correlation with histone methylation of H3K4 (Kappa
= 0.757, P < 0.05) (Table 3).
Silencing efficiency of JARID1B gene by transfection with JARID1B siRNA in Jeko-1 and HL60 cells
After 24 hours transfection with indicated concentration of JARID1B siRNA into Jeko-1 and
HL-60 cells, green fluorescence in the transfected cells were counted by inverted fluorescence microscope, and transfected efficiency
was calculated. Transfection efficiency was
96% ± 3.31% in Jeko-1 (n = 5), and 93% ±
2.56% in HL-60 cells (n = 5) (Figure 2A). The
amplification of JARID1B mRNA attenuated
with concentration dependent manner. Gray
value (to β-actin) showed the amplification of
JARID1B was (1.07 ± 0.15) with 60 nmol/L,
(0.63 ± 0.17) with 120 nmol/L, (0.22 ± 0.7)
with 240 nmol/L respectively in Jeko-1 cells,
compared to control (1.25 ± 0.13), χ² = 12.81,
P < 0.05. Gray value (to β-actin) showed the
amplification of JARID1B was (0.87 ± 0.15) with
30 nmol/L, (0.56 ± 0.09)with 60 nmol/L, (0.12
± 0.03) with 120 nmol/L respectively in HL-60
cells, compared to control (1.13 ± 0.15), χ² =
23.25, P < 0.05 (Figure 2B).
JARID1B siRNA inhibited cell proliferation and
promoted apoptosis in Jeko-1 and HL-60 cells
Depletion of JARID1B significantly suppressed
cell proliferation in Jeko-1 and HL-60 cells. Cells
were transfected with various concentrations
of JARID1B siRNA for 24 hours and cell viability
was measured by MTT assay. Approximately
83.53 ± 4.59% cell proliferation was seen in 15
nmol/L, 79.47 ± 5.88% in 30 nmol/L, 52.33 ±
3.76% in 60 nmol/L, 37.43 ± 4.47% in 120
nmol/L, 19.54 ± 5.18% in 240 nmol/L, 10.35 ±
Figure 5. siRNA JARID1B-induced apoptosis was mitochondria-mediated and caspase dependent. Jeko-1 and HL60 cells were treated respectively for 24 hours with siRNA JARID1B in 0, 30, 60, 120 nmol/L. After siRNA JARID1B
treatment, cytosolic proteins were isolated and separated in 12% SDS gel, transferred onto PVDF membrane and
blotted with BCL-2, procaspase-3, C-myc antibodys. The proteins were determined by immunoblotting using appropriate antibody. β-actin was used as a loading control. A siRNA JARID1B breakup BCL-2, pro-caspase-3 and C-myc
significantly in Jeko-1 cell. B siRNA JARID1B breakup BCL-2, pro-caspase-3 and C-myc in HL-60 cell.
JARID1B modulated apoptosis in Jeko-1 and HL-60 cell lines
179 Int J Clin Exp Pathol 2015;8(1):171-183
4.70% in 480 nmol/L of JARID1B siRNA transfection in Jeko-1 cell line. The effect on HL-60
cells is similar to Jeko-1 cell line. JARID1B siRNA
induced cell viability loss in dose -dependent
manner (Figure 3A). In further experiment, 60
nmol/L of JARID1B siRNA was selected due to
its moderated cytotoxic effect on both cell
lines. After 60 nmol/L transfection, cell proliferation rate was 86.55 ± 5.00%, 52.33 ±
3.15%, 36.47 ± 4.50%, 28.41 ± 9.05%, 19.90
± 5.75% in 12, 24, 36, 48, 60 hours Jeko-1 cell
line, respectively. The effect on HL-60 cells was
similar to Jeko-1 cell line. JARID1B siRNA
induced cell viability loss in a time-dependent
manner (Figure 3B).
To verify if JARID1B siRNA induced cytotoxicity
was due to apoptosis induction, Annexin V-FITC
labeling was performed following 24 hours. In
Jeko-1 cells, Apoptotic rate was 7.16 ± 3.45%,
25.2 ± 4.63%, 52.2 ± 3.49% after transfected
with siRNA JARID1B with 30, 60, 120 nmol/L
for 24 hrs respectively, compared to the
untreated cells (3.57 ± 1.31%), P < 0.05. In
HL-60 cells, Apoptotic rate was 35.2 ± 5.1%,
52.7 ± 3.8%, 62.0 ± 5.7% at the same time
points, compared to the untreated cells (11.0 ±
3.6%), P < 0.05 (Figure 4). The effect on apoptosis was similar in both cell lines. The apoptotic rate was increased in a concentration
dependent manner. We further investigated the
Figure 6. JARID1B siRNA modulated histone methylation and acetylation. The effect of JARID1B siRNA in different
concentration on histone modulation in Jeko-1 and HL-60 cells was determined by immunoblotting. Following 24
hours JARID1B siRNA treatment, total proteins were isolated and separated in 12% SDS gel, transferred onto FVDF
membranes and blotted with JARID1B, Tri-methy-H3K4, Act-H3, Act-H4 and Cyclin-D1. β-actin was used as a loading
control. A. JARID1B siRNA while 30, 60, 120 nmol/L depleted JARID1B, upregulated histone tri-methylated H3K4,
acetylated H3 in concentration-dependent manner in Jeko-1 cell for 24 hours. Alteration was not seen in acetylated
H4. It declined cyclin D1 in concentration-dependent manner in Jeko-1 cell. B. JARID1B siRNA with 30, 60, 120
nmol/L depleted JARID1B, upregulated histone tri-methylated H3K4, acetylated H3 in concentration-dependent
manner in HL-60 cell for 24 hours. Alteration was not seen in acetylated H4.
JARID1B modulated apoptosis in Jeko-1 and HL-60 cell lines
180 Int J Clin Exp Pathol 2015;8(1):171-183
apoptosis associated proteins, BCL-2, pro-caspase-3, and C-myc. It demonstrated that depletion of JARID1B induced breakup of BCL-2, procaspase-3 and C-myc significantly. Depletion of
JARID1B gene with JARID1B siRNA 30, 60, 120
nmol/L for 24 hrs reduced Bcl-2 expression in
0.92 folds, 0.43 folds and 0.14 folds respectively. The expression of procaspase-3 was
reduced 0.52 folds, 0.25 folds, 0.13 folds
respectively. The expression of C-myc was also
reduced by 0.73 folds, 0.63 folds, and 0.22
folds in Jeko-1 cells. It was the same effect in
HL-60 cells (Figure 5).
Depletion of JARID1B gene modulated histone
methylation and acetylation, downregulated
cyclin D1
To test our hypothesis, siRNA JARID1B depleted
JARID1B gene expression, and upregulated histone tri-methylated H3K4 in joke-1 and HL-60
cell lines. The effect on histone lysine methylation was examined by immunocytochemistry
using an antibody against H3K4me3. 30, 60,
120 nmol/L of JARID1B siRNA processing Jeko1 cell for 24 hrs, the expression of JARID1B protein was decreased 0.89 folds, 0.54 folds, 0.21
folds, the expression of histone tri-methylated
H3K4 was increased 2.12 folds, 8.12 folds,
10.24 folds. The effect of siRNA JARID1B on
HL-60 cells was similar to Jeko-1 cells (Figure
6). These data indicate that JARID1B is probably H3K4me3 demethylase. JARID1B siRNA
improved histone acetylation of H3. After 24
hours transfection with JARID1B siRNA in 30,
60, 120 nmol/L, the expression of histone
acetylation of H3 increased 1.18 folds, 1.53
folds, 2.13 folds in HL-60 cells, which is similar
to Jeko-1. But the histone acetylation of H4 was
not changed.
Because MCL is characterized by carrying the
t(11;14)(q13;q32) translocation that leads to
the overexpression of cyclin D1 [8]. We further
study the effect of siRNA JARID1B on cyclin D1
in Jeko-1. The expression of cyclin D1 was
declined after transfection of siRNA JARID1B.
Cyclin D1 was reduced by 0.81 folds, 0.53 folds
and 0.18 folds respectively in indicated concentration of siRNA JARID1B, as compared to
control (Figure 6).
Discussion
JARID1B has been implicated as an oncogene
in breast cancers and prostate [4, 10, 11]. In
2007, the demethylase activity of JARID1B was
evidenced and it was shown to act as a transcriptional repressor in breast cancer, promoting tumor progression by repression of tumor
suppressor genes, including BRCA1 [5].
JARID1B is up-regulated in prostate cancer tissues and associates with androgen receptor
and regulates its transcriptional activity. On the
other hand, a protein described as a JARID1B
isoform, RBP2-H1, is down-regulated in melanomas where it may have tumor suppressive
effects [12]. In this report, we studied 30 cases
of MCL in clinical and pathological diagnosis of
using immunohistochemical method with tissue microarray. We found that JARID1B is higher expression in MCL, H3K4 tri-methylation is
lower expression compared to hyperplastic
lymphadenitis. H3K9 methylation showed no
obvious change. Overexpression of JARID1B
resulted in loss of methyl H3K4. This data is
coincident with our previous study [13], which
showed downreguation of histone methylated
H3K4, histone acetylation of H3, H4 in acute
leukemia. But histone methylated H3K9 is
upregulated. The evidence might prove that
over expression of JARID1B and low expression
of histone tri-methylated H3K4 might involve in
the initiation and development of MCL.
Ki67 antigen is a cell cycle associated nuclear
antigen and is present in all stages of the cell
cycle (G1, S, G2, and M phases) in proliferating
cells, but is absent in G0 and early G1 phases
of cells re-entering the cell cycle [14]. The
monoclonal antibody of Ki67 protein, MIB1,
has been documented useful for the diagnosis
[15, 16] and prognosis [17] of some neoplasms.
In this study, the average index of Ki67 in MCL
is 12.53 ± 23.70%, higher than that in hyperplastic lymphadenitis (1.97 ± 2.70%), P < 0.05.
The result is much similar to the report of
Katzenberger et al. [18] and Gao et al. [19]. We
demonstrated that in addition to prognostic
factor, Ki67 might be a useful marker for distinguishing MCL and hyperplastic lymphadenitis.
The expression of Ki67 has positively correlation with H3K4. It showed that histone methylation of H3K4 might be one of the prognostic
factors in MCL.
Next, we investigated the effects of depletion
of JARID1B on modulation of histone methylated H3K4, histone aceytlation of H3, H4, cyclin
JARID1B modulated apoptosis in Jeko-1 and HL-60 cell lines
181 Int J Clin Exp Pathol 2015;8(1):171-183
D1, improved cell apoptosis and inhibit cell proliferation in Jeko-1 and HL-60 cell lines. We
identified JARID1B as a H3K4 demethylase. It
supported the concept that JARDIB has affect
to tri-methylated H3K4 which is a demethylase
enzyme that in humans is encoded by the
KDM5B gene [20, 21]. It is reported that all
JARID1B target genes are associated with
H3K4me3 and depletion of JARID1B in ESCs
leads to a global increase of H3K4me3 levels
[22]. The results delineate an essential role for
JARDIB-mediated transcriptional control during
ESC differentiation. Our study showed that
depletion of JARID1B improved histone acetylation of H3 in both cell lines. Acetylation and
methylation are the two histone modification
that has been clinically associated with pathological epigenetic disruption in cancer cells. It is
reported that PLU-1/JARID1B has the ability to
physically recruit the class I and class IIa HDACs
direct. In addition, PLU-1/JARID1B can bind to
the transcriptional corepressor N-CoR via an
indirect interaction [23].
In this study, we found that JARID1B siRNA
repressed cyclin D1, which characterized by
98% MCL. Activation of cyclin D1 in these
malignancies is associated with the recruitment of RNA polymerase II to the cyclin D1 promoter and IgH regulatory regions. The cyclin D1
promoter contains a CpG island which can be
potentially regulated by DNA methylation [24].
Suppression of JARID1B resulted in an accumulation of MCF-7 cells in G1 [25]. Cyclin D1 is
suppressed in JARID1B knockdown cells [25].
Cyclin D1 is a therapeutics target for MCL, and
JARDIB might be a new gene therapeutics target for MCL.
It of the he pathogenesis in MCL tnd in This
study demonstrated that depletion of JARID1B
could reduce cell proliferation and induce cell
apoptosis in time and dose-dependent manner
in both jeko-1 and HL-60 cell lines. Depletion of
JARID1B breakup the apoptosis related proteins, procaspase-3, Bcl-2, C-myc, and induced
cell apoptosis. It might contribute to upregulation of tri-methylated H3K4 which is a critical
histone modification regulating transcription.
High levels of H3K4-me3 and H3K4-me2 are
associated with the 5’ regions of virtually all
active genes and are in positive correlation with
transcription rates, active polymerase II occupancy, and histone acetylation [26].
In conclusion, our studies indicated that MCL
has higher JARID1B expression and lower trimethylated H3K4 expression in Depletion of
JARID1B result in suppressed cyclin D1, inhibited cell proliferation and induced cell apoptosis in jeko-1 and HL-60 cell lines. We discovered that the demethylase for H3K4 has equal
effect on the transcription activity inboth cell
lines. Therefore, JARID1B and H3K4 could be a
therapeutic target for MCL and acute myeloid
leukemia.
Acknowledgements
This work was partly supported by grant-in-aid
from the Foundation of Science and Technology
of Fujian Medical University, Fujian, China (No.
FZS08018), Science Research Foundation of
Ministry of Health, United Fujian Provincial
Health, and Education Project for Tackling the
Key Research, P. R. China (WKJ2008-2-55),
Science Research Foundation of Fujian
Province, P. R. China (2012J01420), Medical
Innovations Research Foundation of Fujian
Province, P. R. China (2012-CX-32) and the
major project funded by R & D institutions of
Fujain (2012I2004).
Disclosure of conflict of interest
None.
Address correspondence to: Dr. Xudong Ma,
Department of Hematology, Zhangzhou Affiliated
Hospital of Fujian Medical University, Zhangzhou
363000, Fujian, China. Tel: 0086 596 2082021;
Fax: 0086 596 2593904; E-mail: maxudong005@
hotmail.com
References
[1] Egger G, Liang G, Aparicio A and Jones PA.
Epigenetics in human disease and prospects
for epigenetic therapy. Nature 2004; 429:
457-463.
[2] Lee MG, Norman J, Shilatifard A and
Shiekhattar R. Physical and functional association of a trimethyl H3K4 demethylase and
Ring6a/MBLR, a polycomb-like protein. Cell
2007; 128: 877-887.
[3] Iwase S, Lan F, Bayliss P, de la Torre-Ubieta L,
Huarte M, Qi HH, Whetstine JR, Bonni A,
Roberts TM and Shi Y. The X-linked mental retardation gene SMCX/JARID1C defines a family of histone H3 lysine 4 demethylases. Cell
2007; 128: 1077-1088.
JARID1B modulated apoptosis in Jeko-1 and HL-60 cell lines
182 Int J Clin Exp Pathol 2015;8(1):171-183
[4] Xiang Y, Zhu Z, Han G, Ye X, Xu B, Peng Z, Ma Y,
Yu Y, Lin H, Chen AP and Chen CD. JARID1B is
a histone H3 lysine 4 demethylase up-regulated in prostate cancer. Proc Natl Acad Sci U S A
2007; 104: 19226-19231.
[5] Yamane K, Tateishi K, Klose RJ, Fang J, Fabrizio
LA, Erdjument-Bromage H, Taylor-Papadimitriou
J, Tempst P and Zhang Y. PLU-1 is an H3K4 demethylase involved in transcriptional repression and breast cancer cell proliferation. Mol
Cell 2007; 25: 801-812.
[6] Banck MS, Li S, Nishio H, Wang C, Beutler AS
and Walsh MJ. The ZNF217 oncogene is a candidate organizer of repressive histone modifiers. Epigenetics 2009; 4: 100-106.
[7] Scibetta AG, Santangelo S, Coleman J, Hall D,
Chaplin T, Copier J, Catchpole S, Burchell J and
Taylor-Papadimitriou J. Functional analysis of
the transcription repressor PLU-1/JARID1B.
Mol Cell Biol 2007; 27: 7220-7235.
[8] Sabattini E, Bacci F, Sagramoso C and Pileri
SA. WHO classification of tumours of haematopoietic and lymphoid tissues in 2008: an overview. Pathologica 2010; 102: 83-87.
[9] Ma X, Fang Y, Beklemisheva A, Dai W, Feng J,
Ahmed T, Liu D and Chiao JW. Phenylhexyl isothiocyanate inhibits histone deacetylases and
remodels chromatins to induce growth arrest
in human leukemia cells. Int J Oncol 2006; 28:
1287-1293.
[10] Lu PJ, Sundquist K, Baeckstrom D, Poulsom R,
Hanby A, Meier-Ewert S, Jones T, Mitchell M,
Pitha-Rowe P, Freemont P and TaylorPapadimitriou J. A novel gene (PLU-1) containing highly conserved putative DNA/chromatin
binding motifs is specifically up-regulated in
breast cancer. J Biol Chem 1999; 274: 1563315645.
[11] Barrett A, Madsen B, Copier J, Lu PJ, Cooper L,
Scibetta AG, Burchell J and Taylor-Papadimitriou
J. PLU-1 nuclear protein, which is upregulated
in breast cancer, shows restricted expression
in normal human adult tissues: a new cancer/
testis antigen? Int J Cancer 2002; 101: 581588.
[12] Roesch A, Mueller AM, Stempfl T, Moehle C,
Landthaler M and Vogt T. RBP2-H1/JARID1B is
a transcriptional regulator with a tumor suppressive potential in melanoma cells. Int J
Cancer 2008; 122: 1047-1057.
[13] Ma XD, Huang YQ, Xiao LY and Zou Y. Study on
aberration in histone methylation and acetylation in acute leukemia. Zhonghua Xue Ye Xue
Za Zhi 2010; 31: 523-526.
[14] Gerdes J, Lemke H, Baisch H, Wacker HH,
Schwab U and Stein H. Cell cycle analysis of a
cell proliferation-associated human nuclear
antigen defined by the monoclonal antibody
Ki-67. J Immunol 1984; 133: 1710-1715.
[15] Fujimori Y, Fujimori T, Imura J, Sugai T, Yao T,
Wada R, Ajioka Y and Ohkura Y. An assessment
of the diagnostic criteria for sessile serrated
adenoma/polyps: SSA/Ps using image processing software analysis for Ki67 immunohistochemistry. Diagn Pathol 2012; 7: 59.
[16] Li LX, Crotty KA, McCarthy SW, Palmer AA and
Kril JJ. A zonal comparison of MIB1-Ki67 immunoreactivity in benign and malignant melanocytic lesions. Am J Dermatopathol 2000;
22: 489-495.
[17] Engellau J, Persson A, Bendahl PO, Akerman
M, Domanski HA, Bjerkehagen B, Lilleng P,
Weide J, Rydholm A, Alvegard TA and Nilbert M.
Expression profiling using tissue microarray in
211 malignant fibrous histiocytomas confirms
the prognostic value of Ki-67. Virchows Arch
2004; 445: 224-230.
[18] Katzenberger T, Petzoldt C, Holler S, Mader U,
Kalla J, Adam P, Ott MM, Muller-Hermelink HK,
Rosenwald A and Ott G. The Ki67 proliferation
index is a quantitative indicator of clinical risk
in mantle cell lymphoma. Blood 2006; 107:
3407.
[19] Gao J, Peterson L, Nelson B, Goolsby C and
Chen YH. Immunophenotypic variations in
mantle cell lymphoma. Am J Clin Pathol 2009;
132: 699-706.
[20] Lahoud MH, Ristevski S, Venter DJ, Jermiin LS,
Bertoncello I, Zavarsek S, Hasthorpe S, Drago
J, de Kretser D, Hertzog PJ and Kola I. Gene
targeting of Desrt, a novel ARID class DNAbinding protein, causes growth retardation and
abnormal development of reproductive organs. Genome Res 2001; 11: 1327-1334.
[21] Zhu L, Hu J, Lin D, Whitson R, Itakura K and
Chen Y. Dynamics of the Mrf-2 DNA-binding domain free and in complex with DNA.
Biochemistry 2001; 40: 9142-9150.
[22] Schmitz SU, Albert M, Malatesta M, Morey L,
Johansen JV, Bak M, Tommerup N, Abarrategui
I and Helin K. Jarid1b targets genes regulating
development and is involved in neural differentiation. EMBO J 2011; 30: 4586-4600.
[23] Barrett A, Santangelo S, Tan K, Catchpole S,
Roberts K, Spencer-Dene B, Hall D, Scibetta A,
Burchell J, Verdin E, Freemont P and TaylorPapadimitriou J. Breast cancer associated
transcriptional repressor PLU-1/JARID1B interacts directly with histone deacetylases. Int J
Cancer 2007; 121: 265-275.
[24] Kitazawa S, Kitazawa R and Maeda S.
Transcriptional regulation of rat cyclin D1 gene
by CpG methylation status in promoter region.
J Biol Chem 1999; 274: 28787-28793.
[25] Mitra D, Das PM, Huynh FC and Jones FE.
Jumonji/ARID1 B (JARID1B) protein promotes
breast tumor cell cycle progression through
JARID1B modulated apoptosis in Jeko-1 and HL-60 cell lines
183 Int J Clin Exp Pathol 2015;8(1):171-183
epigenetic repression of microRNA let-7e. J
Biol Chem 2011; 286: 40531-40535.
[26] Ruthenburg AJ, Allis CD and Wysocka J.
Methylation of lysine 4 on histone H3: intricacy
of writing and reading a single epigenetic
mark. Mol Cell 2007; 25: 15-30.

PDF Document reader online

This website is focused on providing document in readable format, online without need to install any type of software on your computer. If you are using thin client, or are not allowed to install document reader of particular type, this application may come in hand for you. Simply upload your document, and Docureader.top will transform it into readable format in a few seconds. Why choose Docureader.top?

  1. Unlimited sharing - you can upload document of any size. If we are able to convert it into readable format, you have it here - saved for later or immediate reading
  2. Cross-platform - no compromised when reading your document. We support most of modern browers without the need of installing any of external plugins. If your device can oper a browser - then you can read any document on it
  3. Simple uploading - no need to register. Just enter your email, title of document and select the file, we do the rest. Once the document is ready for you, you will receive automatic email from us.

Previous 10

Next 10