1. Healthcare

1 MicroRNA181a is overexpressed in T-cell leukemia/lymphoma and related to
2 chemoresistance
3 Zi-Xun YAN M.D.,1,2,* Zhong ZHENG M.D.,1,2,* Wen XUE M.D.,1,2,* Ming-Zhe
4 ZHAO M.D.,3,* Xiao-Chun FEI M.D.,4, Li-Li WU M.D.,4, Li-Min HUANG M.D.,
5 Ph.D,5 Christophe LEBOEUF Ph.D.,6 Anne JANIN M.D., Ph.D.,6 Li WANG M.D.,
6 Ph.D,1,2 Wei-Li ZHAO M.D., Ph.D.1,2
7 *
These authors contributed equally to this manuscript.
1
State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology,
8 9 10 Shanghai Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine,
11 Shanghai, China;
12 Génomique, Laboratory of Molecular Pathology, Shanghai, China; 3 Department of
13 Hematology, Central Hospital of Jin Hua, Zhejiang, China; 4 Department of Pathology,
14 Shanghai Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine,
15 Shanghai, China; 5 Department of Oncology, People's Hospital of Guizhou Province,
16 Guiyang, China;
17 Pathologie, U1165 Inserm, F-75010 France
18 Correspondence: Wei-Li ZHAO, Email: [email protected], Li WANG, Email:
19 [email protected], State Key Laboratory of Medical Genomics, Shanghai
20 Institute of Hematology, Shanghai Rui Jin Hospital, 197 Rui Jin Er Road, Shanghai
21 200025, China, Fax: 0086-21-64743206, Tel: 0086-21-64370045.
2
6
Pôle de Recherches Sino-Français en Science du Vivant et
Université Paris Diderot, Sorbonne Paris Cité, Laboratoire de
22 1 23 Abstract
24 MicroRNAs (miRs) play an important role in tumorogenesis and chemoresistance in
25 lymphoid malignancies. Comparing with reactive hyperplasia, miR181a was
26 overexpressed in 130 patients with T-cell leukemia/lymphoma, including acute T-cell
27 lymphoblastic leukemia (n=32), T-cell lymphoblastic lymphoma (n=16), peripheral
28 T-cell lymphoma, not otherwise specified (n=45), anaplastic large cell lymphoma
29 (n=15) and angioimmunoblastic T-cell lymphoma (n=22). Irrespective to histological
30 subtypes,
31 phosphorylation. In vitro, ectopic expression of miR181a in HEK-293T cells
32 significantly enhanced cell proliferation, activated AKT and conferred cell resistance
33 to doxorubicin. Meanwhile, miR181a expression was upregulated in Jurkat cells,
34 along with AKT activation, during exposure to chemotherapeutic agents regularly
35 applied
36 cyclophosphamide, cytarabine and cisplatin. Isogenic doxorubicin-resistant Jurkat and
37 H9 cells were subsequently developed, which also presented with miR181a
38 overexpression and cross-resistance to cyclophosphamide and cisplatin. Meanwhile,
39 specific inhibition of miR181a enhanced Jurkat and H9 cell sensitivity to
40 chemotherapeutic agents, further indicating that miR181a was involved in acquired
41 chemoresistance. Collectively, miR181a functioned as a biomarker of T-cell
42 leukemia/lymphoma through modulation of AKT pathway. Related to tumor cell
43 chemoresistance, miR181a could be a potential therapeutic target in treating T-cell
44 malignancies.
miR181a
to
T-cell
overexpression
was
leukemia/lymphoma
associated
treatment,
with
such
increased
as
AKT
doxorubicin,
2 45 Key words: miR181a, T-leukemia/lymphoma, chemoresistance.
46 3 47 Introduction
48 Malignancies derived from the T-cell lineages encompass a heterogeneous
49 group of neoplasm. The World Health Organization (WHO) classification recognizes
50 distinctive subtypes of immature T-cell malignancies, like acute T-cell lymphoblastic
51 leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL), as well as mature
52 T-cell malignancies, mainly including peripheral T-cell lymphoma, not otherwise
53 specified
54 angioimmunoblastic T-cell lymphoma (AITL) [1]. Varied from clinicopathological
55 features and biological behavior, they are generally more aggressive than their B-cell
56 counterpart, characterized by resistance to conventional chemotherapy and poor
57 prognosis of the patients [2]. Therefore, biomarkers related to tumor progression and
58 chemoresistance remain to be investigated and may become potential targets for
59 future therapy in T-cell leukemia/lymphoma.
(PTCL-NOS),
anaplastic
large
cell
lymphoma
(ALCL)
and
60 MicroRNAs (miRs), a class of 19- to 23-nucleotide non-coding RNA molecules,
61 regulate gene expression by targeting mRNA at the 3’untranslated region (UTRs)[3].
62 Growing evidences suggested that miRs are critical regulators in tumorogenesis and
63 drug resistance[4, 5]. MiR181 is essential for lymphocyte differentiation and
64 maturation in thymus[6]. More recently, it has been reported that miR181
65 overexpression promotes cell proliferation and activates PI3K/AKT signaling
66 transduction pathway [7, 8]. Activated in lymphoid malignancies[9], AKT plays a
67 pivotal role in tumor progression and resistance to chemotherapeutic agents [10, 11].
68 Here we assessed miR181a expression, as well as its relation to AKT activation and
4 69 chemoresistance in T-cell leukemia/lymphoma.
70 71 Patients and Methods
72 Patients
73 One hundred and thirty patients diagnosed with T-ALL or T-cell lymphoma were
74 enrolled in this study, including 32 T-ALL, 16 T-LBL, 45 PTCL-NOS, 15 ALCL and
75 22 AITL. Histologic diagnoses were established according to the WHO
76 classification.[1] PTCL cases (PTCL-NOS, ALCL and AITL) were treated with
77 CHOP-based chemotherapy. T-LBL and T-ALL cases were treated with
78 HyperCVAD-A/B regimens as previous reported[12, 13]. Response rates were
79 assessed according to the criteria as reported[12, 13]. The clinicopathological data of
80 the patients was listed in Table 1. Thirty-four age and sex-matched cases with reactive
81 hyperplasia were referred as controls. The study was approved by the Institutional
82 Review Board with informed consent obtained in accordance with the Declaration of
83 Helsinki.
84 85 Cell lines and reagents
86 T-leukemia/lymphoma cell lines Jurkat, H9 and embryonic kidney cell line
87 HEK-293T were available from American Type Culture Collection (Manassas, VA,
88 USA). Doxorubicin-resistant Jurkat and H9 cells were established by exposure to
89 gradually increasing concentrations of doxorubicin in vitro, as described by Huang et
90 al.[14]
5 91 92 Cell proliferation assay
93 Cell proliferation was measured by MTT and EdU incorporation assay. Cells
94 were seeded in 96-well plates and incubated with the indicated concentrations of
95 reagents at 37°C. After 72h incubation, 0.1mg of MTT was added to each well and
96 the absorbance was measured at 490nm by spectrophotometry. EdU assay was
97 conducted using Cell-LightTM EdU imaging kit (RiboBio, Guangzhou, China)
98 according to the manufacturer’s instruction.
99 100 MiR181a detection
101 Total RNA was extracted from 20µm-thick paraffin (n=100) or frozen sections
102 (n=64) using RecoverAllTM total nucleic acid isolation kit or Trizol agent following
103 the manufacturer’s protocol. MiR181a expression was analyzed by real-time
104 quantitative RT-PCR using miRNA reverse transcription kit, hsa-miR 181a assay and
105 7500HT Fast Real-time PCR system (Applied Biosystem, CA, USA). RNU24 was
106 used as endogenous control and Jurkat cells for calibration. A relative quantification
107 was calculated using the ΔΔCT method.[15]
108 109 Western blot
110 Cells were lysed in 200µl lysis buffer (0.5M Tris-HCl, pH 6.8, 2mM EDTA, 10%
111 glycerol, 2% SDS and 5% β-mercaptoethanol). Protein extracts (20µg) were
112 electrophoresed on 10% SDS polyacrylamide gels and transferred to nitrocellulose
6 113 membranes. Membranes were blocked with 5% non-fat dried milk in Tris-buffered
114 saline and incubated for 2h at room temperature with appropriate primary antibody,
115 followed
116 immunocomplexes were visualized using chemiluminescence phototope-horseradish
117 peroxidase kit. Actin was used to ensure equivalent protein loading. Antibodies
118 against phosphorylated-AKT (p-AKT), AKT, Actin and chemiluminescence
119 phototope-horseradish-peroxidase kit were obtained from Cell Signaling (Beverly,
120 MA, USA). Anti-PTEN antibody was from Abcam (Cambridge, UK). Horseradish
121 peroxidase-conjugated goat anti-mouse-IgG and goat anti-rabbit-IgG antibodies were
122 from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
by
horseradish
peroxidase-conjugated
secondary
antibody.
The
123 124 Cell transfection
125 HEK-293T cells were incubated with pEZX-181a vector (HmiR0292-MR03) or
126 a control vector pEZX-ct (CmiR0001-MR03, Genecopia, MD, USA) and
127 lipofectamine 2000 (Invitorgen, CA, USA) for 24h and replaced in fresh medium for
128 further experiments. To inhibit miR181a expression, Jurkat and H9 cells were
129 transfected with 10nM antagomir using lipofectamine 2000 (Invitorgen) for 24h. The
130 miR181a antagomir and the negative control were synthesized by Shanghai Biotend
131 Biotechnologies Co., Ltd (Shanghai, China).
132 133 Immunohistochemistry assay
Immunohistochemistry was performed on 5µm-paraffin sections with an indirect
134 7 135 immunoperoxidase method using antibodies against p-AKT (Cell Signaling).
136 Expression levels were scored semi-quantitatively based on percentage of positive
137 cells: +, <25%; ++, 25-49%; +++, 50-74%; ++++, 75-100%.
138 139 Statistical analysis
140 Differences of miR181a expression among groups were assessed using
141 Mann-Whitney U test. The association between miR181a and p-AKT expression in
142 human tumor samples was analyzed by Fisher’s exact test. In vitro experimental
143 results were expressed as mean±S.D. of data obtained from three separate experiments
144 and determined using t-test to compare variance. P<0.05 was considered statistically
145 significant.
146 147 Results
148 MiR181a was overexpressed in T-cell leukmia/lymphoma and related to AKT
149 activation.
150 Compared with reactive hyperplasia, miR181a was overexpressed in T-cell
151 leukemia/lymphoma (P<0.0001, Figure 1A). No significant difference was observed
152 among T-ALL and subtypes of T-cell lymphoma (P=0.5153, Figure 1B).
153 The median value of relative miR181a expression in T-cell leukemia/lymphoma
154 was 2136. The patients with miR181a expression level over and equal to the median
155 value were regarded as high miR181a expression, whereas those below the median
156 value were included in the low miR181a expression. Patients with high miR181a
8 157 expression had significantly lower overall response rate (ORR) than those with low
158 miR181a
159 immunohistochemistry in primary tumor sections of 12 T-cell lymphoma patients (6
160 cases from high miR181a expression group and 6 cases from low miR181a expression
161 group, Figure 2A). High miR181a expression was associated with increased positivity
162 of p-AKT (P=0.0152, Figure 2B).
expression
(Table
1).
P-AKT
expression
was
detected
by
163 164 MiR181a promoted cell proliferation and induced chemoresistance through
165 activating AKT.
166 T-leukemia/lymphoma cell lines Jurkat and H9 possessed higher levels of
167 miR181a expression than that of HEK-293T cells (P=0.0023 and P=0.0030,
168 respectively, Figure 3A). To gain insight into the biological function of miR181a,
169 HEK-293T cells, with lowest miR181a expression, were transiently transfected with
170 miR181a (pEZX-181a, Figure 3B). Ectopic expression of miR181a remarkably
171 accelerated cell growth, as compared to the control cells (pEZX-ct). In parallel with
172 increased cell proliferation, the percentage of EdU-positive cells was significantly
173 higher in pEZX-181a cells (52.7%±8.7%) than in pEZX-ct cells (20.7%±7.0%,
174 P=0.0458, Figure 3C). Of note, overexpression of miR181a increased AKT
175 phosphorylation, while the total protein level remained constant (Figure 3D). AKT is
176 the key regulator of cell proliferation and drug resistance.[11, 16] Accordingly, the
177 IC50 of doxorubicin was significantly increased in the miR181a-overexpressing
178 HEK-293T cells, as compared to the control cells (21.3±3.1 nM vs 9.8±2.3 nM,
9 179 P=0.0260, Figure 3E).
180 181 MiR181a
overexpression
182 T-leukemia/lymphoma cells.
corresponded
to
chemoresistance
in
183 Doxorubicin (DOX), cyclophosphamide (CTX), cytarabine (Ara-C) and
184 cisplatin are main chemotherapeutic agents used in treating T-cell malignancies.
185 When Jurkat cells were exposed to these agents for 48h, miR181a expression was
186 significantly increased (P=0.0019, P=0.0016, P=0.0172, and P<0.0001, respectively,
187 Figure 4A), in according with increased AKT phosphorylation detected by Western
188 blot (Figure 4B).
189 Acquired drug resistance is an important obstacle that impairs the success of
190 cancer treatment. Isogenic doxorubicin-resistant sublines were developed as
191 previously reported [14, 17], at the concentrations of 7.5nM (Jurkat/7.5nM DOX) and
192 15nM (Jurkat/15nM DOX) in Jurkat cells, as well as 5nM (H9/5nM DOX) and 10nM
193 (H9/10nM DOX) in H9 cells. Compared with the parental cells (Jurkat cells, 44.3±
194 4.0 nM, H9 cells, 14.7±5.5 nM), IC50 of doxorubicin was significantly increased in
195 Jurkat/7.5nM DOX and Jurkat/15nM DOX cells (70.1±8.0 nM and 100.0±8.0 nM,
196 P=0.0453 and P=0.0152, respectively, Figure 4C), and in H9/5nM DOX and
197 H9/10nM DOX cells (28.0±4.3 nM and 41.3±4.7 nM, P=0.0481 and P=0.0443,
198 respectively, Figure 4D). Accordingly, levels of miR181a expression were
199 significantly higher in doxorubicin-resistant cells than in the parental cells (1.8±0.2
200 fold in Jurkat/7.5nM DOX and 2.8±0.3 fold in Jurkat/15nM DOX cells, P=0.0253
10 201 and P=0.0112, Figure 4E, 2.1±0.6 fold in H9/5nM and 3.4±0.2 fold in H9/10nM
202 DOX cells, P=0.0384 and P=0.0032, respectively, Figure 4F), consistent with AKT
203 activation (Figure 4G and 4H). Increased miR181a expression was linked to AKT
204 phosphorylation, not only in PTEN-negative Jurkat cells[18], but also in
205 PTEN-positive H9 cells (Figure 4G and 4H). Therefore, regulation of AKT
206 phosphorylation
207 leukemia/lymphoma. Further drug-sensitivity test showed that these resistant sublines
208 with miR181a overexpression also had cross-resistance to other chemotherapeutic
209 agents (Table 2). Therefore, exposure to chemotherapeutic agents could induce
210 miR181a expression and AKT activation, which is closely related to acquired
211 chemoresistance.
could
be
independent
on
PTEN
expression
in
T-cell
212 213 MiR181a
inhibition
enhanced
214 chemotherapeutic agents
T-leukemia/lymphoma
cell
sensitivity
to
215 Specific inhibition of miR181a in Jurkat and H9 cells as well as their resistant
216 sublines, using an antagomir, significantly increased cell sensitivity to doxorubicin
217 (Figure 5A and 5B) and decreased AKT phosphorylation (Figure 5C and 5D). Similar
218 as doxorubicin, decreased miR181a expression was also related to reduced IC50 of
219 cisplatin and cyclophosphamide in doxorubicin-resistant Jurkat and H9 cells (Table
220 3).
221 222 Discussion
11 223 In addition to genetic abnormalities, epigenetic aberrations, particularly those of
224 miRs, participate in human carcinogenesis [5, 19]. MiR181a is critically involved in
225 hematological malignancies. In acute myelogenous leukemia, increased miR181a
226 expression was significantly associated with a higher complete remission rate of the
227 patients[20]. However, as recently reported in lymphoid malignancies like acute
228 lymphoblastic leukemia and multiple myeloma, miR181 overexpression was related
229 to advanced stage and tumor progression[7, 21]. In according with malignant T cells,
230 miR181a is upregulated in normal T-cell counterpart and miR181a/b-deficient mice
231 show severe defects in T-cell development[6, 8]. Therefore, miR181 may have
232 different roles in hematological malignancies. Our study showed that miR181a,
233 independent on histological subtypes, was overexpressed and these patients were less
234 responded to treatment, referring miR181a as a common biomarker of
235 chemoresistance in T-cell leukemia/lymphoma.
236 Chemoresistance, determining therapeutic effect and clinical outcome of the
237 patients, is one of the control factors in cancer treatment, including T-cell
238 leukemia/lymphoma [22]. Here miR181a was closely related to chemoresistance in
239 T-leukemia/lymphoma. This was consistent with previous reports in B-cell lymphoma
240 that high expression of miR181 could lead to decreasing proapoptotic protein Bim and
241 increasing resistance to chemotherapy [23]. AKT is a key tuning point in tumor cell
242 growth and chemosensitivity [24, 25]. MiR181 is a central regulator of PI3K pathway,
243 since miR181a/b-deficient mice showed severe defects in lymphoid development and
244 T-cell homeostasis associated with impaired PI3K/AKT cascade [8]. As mechanism
12 245 of action, miR181 targets PTPN, DUSP5, DUSP6, resulting in PI3K/AKT activation
246 and tumorogenesis in murine T-cell leukemia [7, 8]. Our results showed that ectopic
247 expression of miR181a leads to AKT phosphorylation, enhancing cell proliferation
248 and inducing cell resistance to chemotherapy in T-cell leukemia/lymphoma. This
249 correlation of miR181a overexpression with AKT activation was not only observed in
250 cell lines, but also in primary tumor samples of patients with T-leukemia/lymphoma.
251 Apart from primary chemoresistance, acquired chemoresistance is also an important
252 factor of treatment failure. MiR181a expression of Jurkat cells was significantly
253 upregulated after exposure to chemotherapeutic agents and linked to increased AKT
254 phosphorylation. Meanwhile, isogenic doxorubicin-resistant cell lines were developed,
255 which
256 chemotherapeutic drugs. The relative resistance to chemotherapeutic agents was along
257 with increased miR181a expression and subsequent AKT activation, further
258 confirming that miR181a induced AKT activation and contributed to chemoresistance
259 in T-cell leukemia/lymphoma.
were
resistant
to
doxorubicin
and
had
cross-resistance
to
other
260 261 Conclusions
262 MiR181a was involved in T-cell leukemia/lymphoma through modulation of
263 AKT pathway. Functioned as a critical regulator of chemosensitivity, miR181a could
264 thus be a promising therapeutic target in treating T-cell malignancies resistant to
265 chemotherapy.
266 13 267 Conflicts of Interest
268 The authors declare no conflict of interest.
269 270 Acknowledgements
271 This work was supported, in part, by the National Natural Science Foundation of
272 China (81172254, 81101793 and 81325003), Innovation Fund Projects of Shanghai
273 Jiao Tong University (BXJ201312), the Shanghai Commission of Science and
274 Technology (11JC1407300, 14430723400, and 14140903100), and the Program of
275 Shanghai Subject Chief Scientists (13XD1402700).
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360 361 362 17 363 Figure Legends
364 Figure 1. MiR181a was overexpresssed in T-cell leukemia/lymphoma.
365 A. As detected by real-time quantitative PCR, miR181a was overexpressed in T-cell
366 malignancies. ***, P<0.001 comparing with reactive hyperplasia. The relative
367 expression level of each patient was calculated based on the lowest expression value.
368 B. MiR181a was overexpressed in acute T-cell lymphoblastic leukemia (T-ALL,
369 n=32), as well as subtypes of T-cell lymphoma, including T-cell lymphoblastic
370 lymphoma (T-LBL, n=16), peripheral T-cell lymphoma, not otherwise specified
371 (PTCL-NOS, n=45), anaplastic large cell lymphoma (ALCL, n=15), and
372 angioimmunoblastic T-cell lymphoma (AITL, n=22).
373 374 Figure 2. MiR181a overexpression was related to AKT activation in T-cell
375 leukemia/lymphoma.
376 As revealed by immunohistochemistry (A), increased positivity of p-AKT was
377 observed in primary tumor samples of T-cell lymphoma patients with high miR181a
378 expression (n=6), compared to those with low miR181a expression (n=6) (B). *,
379 P<0.05 comparing with low miR181a expression. Bar=50 µm.
380 381 Figure 3. Ectopic expression of miR181a enhanced cell proliferation and
382 resistance to doxorubicin through AKT activation.
383 A. T-leukemia/lymphoma Jurkat and H9 cells had significantly higher expression
384 levels of miR181a than that of HEK-293T cells. **, P<0.01 comparing with
18 385 HEK-293T cells. B. Transfection with miR181a (pEZX-181a) in HEK-293T cells
386 resulted in significantly increased miR181a expression. **, P<0.01 comparing with
387 the control pEZX-ct cells. C. EdU incorporation assay in HEK-293T cells showed that
388 miR181a-overexpressing pEZX-181a cells presented with increased EdU-positive
389 cells. *, P<0.05, comparing with the control pEZX-ct cells. Bar=20µm. D.
390 Overexpression of miR181a increased AKT phosphorylation, while the total protein
391 level remained constant. E. IC50 of doxorubicin was significantly higher in the
392 pEZX-181a cells than in the control pEZX-ct cells. *, P<0.05 comparing with the
393 control pEZX-ct cells.
394 395 Figure 4. Exposure of T-leukemia/lymphoma cells to chemotherapeutic agents
396 upregulated miR181a expression and resulted in AKT activation.
397 A. When Jurkat cells were treated with chemotherapeutic agents for 48h, miR181a
398 expression was significantly increased. CON, untreated; DOX, doxorubicin; CTX,
399 cyclophosphamide; Ara-C, cytarabine. *, P<0.05, **, P<0.01; ***, P<0.001
400 comparing with the CON cells. B. In accordance with miR181a upregulation,
401 increased AKT phosphorylation was observed by western blot, while the total protein
402 level remained constant. C and D. IC50 of doxorubicin was significantly increased in
403 doxorubicin-resistant Jurkat (C) and H9 (D) cells. E and F. MiR181a expression was
404 significantly increased in doxorubicin-resistant Jurkat (E) and H9 (F) cells. CON,
405 untreated, *, P<0.05, **, P<0.01 comparing with the CON cells. G and H. P-AKT
406 expression was significantly increased in doxorubicin-resistant Jurkat (G) and H9 (H)
19 407 cells.
408 409 Figure
410 T-leukemia/lymphoma cells and resistant sublines sensitivity to doxorubicin,
411 along with p-AKT downregulation.
412 A and B. IC50 of doxorubicin was significantly decreased in antagomir-treated Jurkat
413 (A) and H9 (B) cells. **, P<0.01, ***, P<0.001 comparing with the control cells. C
414 and D: Decreased AKT phosphorylation was found in miR181a antagomir-treated
415 Jurkat (C) and H9 (D) cells.
5.
Speicific
inhibition
of
miR181a
expression
could
increase
20 Figure 1
A
B
MiR181a expression
MiR181a expression
p -A K T e xp re ssio n
100
80
60
40
20
0
TL
AI
L
C
AL
66 %
83 %
17 %
17 %
p-AKT
positive cells
++++
+++
++
+
Lo
w
h
m
1a
1a
18
18
iR
iR
m
S
17 %
ig
Low miR181a
n=6
LN
TL
PT
C
*
H
High miR181a
n=6
O
LL
Figure 2
B
Patients (%)
A
Re a ctive
h yp e rp la sia
n =3 4
TA
T-ce ll
ma lig n a n cie s
n =1 3 0
BL
Relative
miR181a levels
Relative
miR181a levels
***
21 Figure 3
A
B
HE K -2 9 3 T
20
**
**
10
0
HE K -2 9 3 T H9
C
0
p E Z X - ct p E Z X- 1 8 1a
Ju rka t
Ove rla y
pEZ X - 181a pE ZX - ct
EdU
E d U p o sitive ce lls
pEZX- 181a pEZ X - ct
D
HE K -2 9 3 T
E
50
AKT
β - a ctin
IC50 ( nM)
p -A K T
*
0
50
100
Percentage( % )
HE K 2 9 3 T
p E Z X - ct p E Z X- 1 8 1a
10
E d U in co rp o ra tio n a ssa y
DA P I
**
20
Relative
miR18 1a levels
Relative
miR18 1a levels
MiR1 8 1a e xp re ssio n in ce ll lin e s
*
25
0
p E Z X - ct p E Z X- 1 8 1a
22 Figure 4
B
Relative
miR181a levels
**
8
A
at
C
is
pl
-C
ra
TX
O
O
X
N
***
C
**
*
C
12
Ju rka t
in
Ju rka t
MiR1 8 1 a e x p r e s s i o n ( 4 8 h )
D
A
p -A K T
AKT
4
β-actin
0
CON
C
DOX
CT X
A ra -C Cisp la tin
D
G
IC50 in H9
Ju rka t
ol
2
X
X
DO
ol
on
tr
**
*
O
X
H9
C
4
p-AKT
2
PTEN
0
DO
X
X
15
nM
75
nM
DO
ol
tr
on
nM
15
nM
75
C
DO
X
DO
ol
tr
on
X
β-actin
C
nM
nM
M
10
H
MiR181a in H9
Relative
miR181a levels
*
*
β-actin
D
F
MiR181a in Jurkat
4
DO
ol
C
5n
75
15
nM
nM
on
tr
DO
DO
X
ol
tr
on
C
X
0
E
15
PTEN
nM
0
Relative
miR181a levels
tr
p-AKT
25
10
75
on
*
*
*
50
C
*
IC50 (nM)
IC50 (nM)
150
D
O
X
IC50 in Jurkat
23 Figure 5
A
IC 50 in Jurkat
Jurkat
**
DOX-resistant Jurkat
***
150
100
50
0
H9
150
DOX-resistant H9
** *
**
100
50
D
β - a c tin
β - a c tin
ag
l
p -A K T
An
t
tro
on
C
p -A K T
om
ir
H9
om
ir
ag
An
t
on
tro
l
J u rk a t
C
An
ta
go
m
ir
10
nM
D
O
X
An
ta
go
m
ir
C
on
tro
l
An
ta
go
m
ir
15
nM
D
O
X
An
ta
go
m
ir
C
on
tro
l
0
C
IC 50 in H9
IC 50 (nM)
IC 50 (nM)
200
B
24 Table 1 Clinicopathological characteristics of 130 patients with T-cell leukemia/lymphoma
High miR181a
Low miR181a
expression
Expression
(n=65)
(n=65)
≥60
13
17
<60
52
48
Male
42
32
Female
23
33
Normal
19
23
Above normal
46
42
T-ALL
15
17
T-LBL
8
8
PTCL-NOS
22
23
ALCL
6
9
AITL
14
8
Characteristics
P value
Age (years)
0.5328
Gender
0.1106
Lactic dehydrogenase level (LDH)
0.5740
Pathological subtypes
0.6656
International prognostic index (IPI)
Low
12
11
Low/intermediate
23
33
Intermediate/high
20
15
High
10
6
38
50
Overall response (CR+PR)
0.3152
0.0385
* CR, complete remission; PR, partial remission
Table 2 Cross-resistance of doxorubicin-resistant cells to other chemotherapeutic agents
IC50
Agents
Jurkat
IC50
Doxorubicin
resistant-Jurkat
P value
H9
Doxorubicin
resistant-H9
P value
Cisplatin (uM)
6.2± 0.3
9.0±0.4
0.0039
5.7±0.3
8.5± 0.5
0.0067
Cyclophosphamide (mM)
3.3±0.2
4.8±0.2
0.0056
2.4±0.2
3.9±0.3
0.0106
25 (mM)
Cyclophosphamid
(uM)
Cisplatin
Agents
3.3±0.2
1.6±0.2
5.1±0.1
Antagomir
Control
6.6±0.2
Jurkat
Jurkat
IC50
0.0018
0.0014
Value
P
4.8±0.2
3.6±0.2
7.0±0.4
t Antagomir
Control
9.0±0.4
resistant-Jurka
Doxorubicin
resistant-Jurkat
Doxorubicin
IC50
0.0214
0.0215
Value
P
2.4±0.2
5.7±0.3
Control
H9
H9
0.9±0.1
4.4±0.2
Antagomir
IC50
0.0019
0.0264
Value
P
3.9±0.3
8.5±0.5
Control
resistant-H9
2.5±0.2
6.0±0.5
Antagomir
resistant-H9
Doxorubicin
IC50
Doxorubicin
Table 3 Inhibition miR181a expression sensitized T-leukemia/lymphoma cells to chemotherapeutic agents
0.0134
0.0191
Value
P
26