# miR-146a

Authors: Lan Xu; Hua Zhong; Haixia Wan; Fang-yuan Chen; Jihua Zhong; Fei Xiao; Jia Liu; Lijing Shen
Journal: Disease Markers
 (2014)

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## Abstract

Background. Although the curative rate for acute promyelocytic leukemia (APL) has been improved over decades, long-term prognosis is still poor. The genetic pathways that regulated cell lineage fate during the development of APL remain unclear.Methods. We investigated the correlations ofmiR-146a expression with its target gene Smad4 and the biological behaviors of NB4 cells. We also analyzed their expression in clinical samples from APL patients.Results. miR-146a influenced apoptosis and proliferation in NB4 cells. miR-146a influenced endogenous Smad4 protein levels in APL cells. miR-146a expression levels were positively correlated with white cell counts and PML/RARα fusion protein expression. miR-146a expression levels were negatively correlated with Smad4 protein and the helper T cell (Th)/the suppressor T cell (Ts) ratio in these patients. Conclusions. These findings indicated that miR-146a played an important role in the development of APL in part through the repression on Smad4 protein expression. miR-146a functioned as an oncogene and may be a novel prognostic biomarker in APL.


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## Body

## 1. Introduction

Acute promyelocytic leukemia (APL) has been identified as an M3 subtype of acute myelogenous leukemia (AML) by the French-American-British (FAB) classification. APL is characterized by maturation arrest at the promyelocytic stage [1]. A specific t (15; 17) chromosomal translocation encodes a promyelocytic leukemia (PML) and retinoic acid receptor-α (RARα) fusion protein to form PML/RARα, an oncogenic protein found in approximately 10%–25% of adults with AML [2]. PML/RARα interferes with the process of myeloid differentiation by repressing the transcription of retinoid acid- (RA-) responsive genes.Although the outcomes of APL have been dramatically improved since the successful introduction of all-trans retinoic acid (ATRA), arsenic acid, and combined anthracycline-based chemotherapy, more than 10% of newly diagnosed APL patients die of the disease; moreover, the 5-year cumulative incidence of relapse is around 15% in high-risk subgroups [3]. Furthermore, the specific genes and pathways that regulate lineage fate during APL development remain unclear.MicroRNAs (miRNAs) are a group of highly conserved non-protein-coding RNAs comprised of about 19–25 nucleotides. miRNAs can regulate the expression of a variety of genes by binding the 3′ untranslated regions (3′ UTRs) of messenger RNAs (mRNAs) in a sequence-specific manner to regulate mRNA translation or degradation in eukaryotic cells [4], ultimately affecting cell proliferation, apoptosis, development, and differentiation [5].miR-146a was first identified as having a role in the innate immune and inflammatory response to microbial infection [6]. Later studies showed thatmiR-146a is expressed at relatively high levels in bone marrow (BM) CD34+ progenitors from healthy donors but is found at low levels in AML patients and even lower levels in monocytes, granulocytes, erythrocytes, and megakaryocytes from the peripheral blood or BM of healthy donors [7]. miR-146a is strongly upregulated during megakaryopoiesis in mice [8], and dysregulation ofmiR-146a has been found in APL cells following retinoic acid (RA) induction [9–12]. Our previous study confirmed the reduced expression ofmiR-146a in NB4 cells following treatment with ATRA [13]. However, the role ofmiR-146a in the clinical progression of APL remains unknown.In this study, we investigated the correlations ofmiR-146a expression with the expression of its target gene Smad4 and the biological behaviors of NB4 cells, such as proliferation and apoptosis. To further elucidate the role ofmiR-146a in APL, we analyzed its expression in samples from 32 APL patients for whom clinical data were available. Interestingly, the expression levels ofmiR-146a were positively correlated with white blood cell (WBC) counts in the peripheral blood and expression of the PML/RARα fusion protein. miR-146a expression levels were also negatively correlated with Smad4 protein and the helper T cell (Th)/the suppressor T cell (Ts) ratio in these patients. These results indicated thatmiR-146a functioned as an oncogene in APL and may be a potential biomarker for malignancy.


## 2. Materials and Methods

### 2.1. Cell Culture and Transient Transfection withmiR-146a Mimics

The human promyelocytic cell line NB4 was a gift from Shanghai Institution of Hematology and was cultured in RMPI 1640 medium with 10% fetal calf serum (FCS; Gibco, BRL, UK) at 37°C in a 5% CO2 humidified incubator. On the day of transfection, 5 × 106 cells/mL were plated in 6-well plates with RMPI 1640 supplemented with 10% FBS. Transfections were carried out with 100 nM DMEM-diluted pre-miRmiR-146a precursor, pre-miR negative control, anti-pre-miRmiR-146a precursor, or anti-pre-miR negative control (Ambion, Carlsbad, CA, USA) using siPORT NeoFX Transfection Agent (Ambion, Carlsbad, CA, USA). Twenty-four hours after transfection, the medium was replaced, and the cells were cultured for another 24 h. To monitor the transfection efficiency of the miRNAs, pre-miRhas-miR-1 precursor and anti-miRhas-let-7c miRNA inhibitor were transfected into NB4 cells in parallel experiments according to the manufacturer’s instructions.


### 2.2. Cell Proliferation, Apoptosis, and Cell Cycle Analyses

Live cell proliferation assays were carried out by trypan blue dye exclusion using a Bio-Rad automatic cell counter (Bio-Rad, Berkeley, CA, USA). The number of NB4 cells was calculated in triplicate after 24 or 48 h of culture.Apoptotic cells were detected with an Alexa Fluor 488 annexin V/Dead cell apoptosis kit (Invitrogen, Carlsbad, CA, USA) by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). Early apoptotic cells were defined as Annexin-V-positive/propidium iodide- (PI-) negative cells. The experiments were repeated 3 times.For analysis of the cell cycle distribution, NB4 cells were washed 3 times with cold phosphate-buffered saline (PBS), fixed with 70% ethanol, and incubated at −20°C for more than 12 h. Before examination, the cells were washed with cold PBS and stained with 0.5 mL PI staining buffer (200 mg/mL RNase A and 50 μg/mL PI in PBS). The mixture was incubated at 37°C for 30 min in the dark, and cell cycle distribution was analyzed by flow cytometry (BD Biosciences). The experiments were repeated 3 times.


### 2.3. miRNA and mRNA Extraction and Real-Time Quantitative Polymerase Chain Reaction (RTq-PCR)

miRNA was extracted with a mirVana miRNA isolation kit (AM1560, Applied Biosystems, Foster City, CA, USA). MaturemiR-146a was detected by TaqMan miRNA assay as previously reported [13], using a TaqMan_MicroRNA Reverse Transcription Kit (4366597, Applied Biosystems) and PCR 9700 sequence detection system (Applied Biosystems). U48 was used as an internal control. miRNA levels were expressed as relative 2
-
Δ
CT values.mRNAs of other genes (Smad4,PML/RARα,ABL, andGAPDH), excluding miRNAs, were extracted using a QIAampRNA blood mini kit (Qiagen, Valencia, CA, USA), and 10 μg mRNA was used in each reverse transcription reaction, carried out with Superscript II reverse transcriptase and random primers (Invitrogen). Gene expression was determined by RTq-PCR as previously described [14] on a PCR 9700 sequence detection system.ABL andGAPDH were used as internal controls. mRNA levels were expressed as relative 2
-
Δ
CT values.


### 2.4. Western Blot Analysis

A total of 5 × 106 cells of each experimental group were harvested and subsequently were washed twice with phosphate-buffered saline (PBS). Cells were then lysed with lysis buffer (300 mM NaCl, 0.5% NP-40, 1 mM DTT, 200 mM PMSF, protease inhibitor tablet) for 20 minutes. The protein concentration of the cell lysates was quantified using a bicinchoninic acid (BCA) protein Assay Kit (Kangchen, China). 20 μg protein extracted from each experimental sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes and blotted with rabbit polyclonal anti-Smad4 (1 : 5000, Cell Signaling Technology, Danvers, MA, USA) or rabbit monoclonal anti-GAPDH (1 : 2000, Cell Signaling Technology) antibodies. Protein bands were visualized with the use of enhanced chemiluminescence reagent (Pierce), according to the manufacturer’s instructions, and band intensities were analyzed using Bandscan 5.0 (Glyko, Hayward, CA, USA).


### 2.5. Patients, Sample Collection, and Therapeutic Methods

Thirty-two APL patients and ten iron deficiency anemia patients as control who attended Renji Hospital were enrolled in this study. The clinical characteristics of these patients are shown in Table1. Bone marrow was collected from patients at diagnosis or after therapy. APL patients were treated with ATRA (30 mg/m2) and daunorubicin (DNR; 60 mg/m2)/idarubicin (IDA; 8 mg/m2) daily for 3 days. Arsenic trioxide was used at 10 mg daily until patients achieved complete remission. The details of these patients including treatment protocol can be found in Table 2. Protein extracts from 14 patients were used for western blotting, including 6 pairs of samples taken before and after therapy. Written informed consent for participation in this study was obtained from all patients. The study was approved by the Ethics Committee of Renji Hospital (number 81270626).Table 1
Clinical characteristics of APL patients.
Characteristic
Mean ± SD
Age
42.22 ± 2.82
Gender (male/female)
13/19
WBC count (109/L)
12.22 ± 3.45
BM blast cell percentage (%)
84.70 ± 1.89
PML/RARα
419.81 ± 48.26
Th/Ts ratio
1.48 ± 0.10Table 2
Clinical variables for controls and APL patients.
Patients ID
Gender
Age
WBC (×109/L)
PML/RARa : ABL relative ratio
Th/Ts
Smad4/GAPDH
Therapy
Control
C1
M
44
6.32
NA
0.98
NA
NA
C2
F
34
4.67
NA
1.03
NA
NA
C3
F
59
5.96
NA
0.61
NA
NA
C4
F
28
11.30
NA
1.31
NA
NA
C5
M
24
8.35
NA
1.87
NA
NA
C6
F
63
5.90
NA
1.55
NA
NA
C7
F
30
7.40
NA
1.07
NA
NA
C8
M
38
3.76
NA
1.12
NA
NA
C9
F
68
7.75
NA
2.05
NA
NA
C10
M
35
4.81
NA
2.11
NA
NA
APL
A1
F
53
0.80
174
2.03
1.4061
ATRA + ATO + DNR
A2
F
56
3.20
285
1.69
1.2696
ATRA + ATO + IDA
A3
F
67
22.40
713.5
1.01
0.7683
ATRA + ATO + DNR
A4
M
33
6.56
493.5
1.49
0.9575
ATRA + ATO + IDA
A5
M
76
57.44
829.5
0.57
0.3519
ATRA + DNR
A6
F
33
7.50
537
1.39
NA
ATRA + DNR
A7
M
64
1.50
169
1.85
1.7688
ATRA + ATO + IDA
A8
F
44
2.30
416.5
1.88
1.3570
ATRA + ATO + IDA
A9
F
34
3.80
434
1.72
NA
ATRA + ATO + IDA
A10
F
63
6.30
501.5
0.97
1.1272
ATRA + ATO + IDA
A11
M
28
64.60
1048
0.36
0.2464
ATRA
A12
F
24
1.05
225.5
1.95
NA
ATRA + ATO + IDA
A13
F
24
1.60
143
1.8
2.0794
ATRA + ATO + IDA
A14
M
30
7.60
221
1.61
0.8966
ATRA + ATO + IDA
A15
F
38
34.10
617
0.49
NA
ATRA + DNR
A16
M
21
2.30
378
1.15
NA
ATRA + ATO + DNR
A17
F
23
0.70
183
2
1.5293
ATRA + DNR
A18
M
48
2.60
331
1.27
NA
ATRA + DNR
A19
F
24
3.90
411
1.86
NA
ATRA + ATO + IDA
A20
F
34
9.70
612
1.31
NA
ATRA + ATO + DNR
A21
F
60
1.30
173
1.87
1.9294
ATRA + ATO + IDA
A22
F
50
1.60
199
1.91
NA
ATRA + ATO + IDA
A23
M
30
1.60
189
1.85
NA
ATRA + ATO + IDA
A24
F
56
1.10
191
1.99
NA
ATRA + ATO + IDA
A25
M
51
3.20
367
1.7
NA
ATRA + ATO + IDA
A26
F
24
2.20
336
1.91
NA
ATRA + ATO + IDA
A27
M
40
1.00
156
1.97
NA
ATRA + ATO + IDA
A28
M
15
28.30
729
0.93
NA
ATRA + ATO + IDA
A29
F
62
0.60
201
2.13
NA
ATRA + ATO + DNR
A30
M
50
41.40
854
0.64
NA
ATRA + ATO + IDA
A31
F
44
67.20
1137
0.3
0.3373
ATRA + ATO + IDA
A32
M
52
1.50
179
1.82
NA
ATRA + ATO + IDA
M: male; F: female; WBC: white blood cell; ATRA:all  trans retinoic acid; ATO: arsenic trioxide; DNR: daunorubicin; IDA: idarubicin; NA: not available.


### 2.6. Statistical Analyses

To investigate whethermiR-146a expression correlated with clinical quantitative variables (e.g., WBC counts), we used Pearson correlation analysis within SPSS 16.0 (SPSS Inc., Chicago, IL, USA). The Mann-Whitney U test was carried out to assess the differential expression ofmiR-146a for statistical significance. Data from western blotting were analyzed by paired t-tests using SAS version 12.6. All results were presented as the mean ± standard deviation (SD). P values of less than 0.05 were considered statistically significant.


## 2.1. Cell Culture and Transient Transfection withmiR-146a Mimics

The human promyelocytic cell line NB4 was a gift from Shanghai Institution of Hematology and was cultured in RMPI 1640 medium with 10% fetal calf serum (FCS; Gibco, BRL, UK) at 37°C in a 5% CO2 humidified incubator. On the day of transfection, 5 × 106 cells/mL were plated in 6-well plates with RMPI 1640 supplemented with 10% FBS. Transfections were carried out with 100 nM DMEM-diluted pre-miRmiR-146a precursor, pre-miR negative control, anti-pre-miRmiR-146a precursor, or anti-pre-miR negative control (Ambion, Carlsbad, CA, USA) using siPORT NeoFX Transfection Agent (Ambion, Carlsbad, CA, USA). Twenty-four hours after transfection, the medium was replaced, and the cells were cultured for another 24 h. To monitor the transfection efficiency of the miRNAs, pre-miRhas-miR-1 precursor and anti-miRhas-let-7c miRNA inhibitor were transfected into NB4 cells in parallel experiments according to the manufacturer’s instructions.


## 2.2. Cell Proliferation, Apoptosis, and Cell Cycle Analyses

Live cell proliferation assays were carried out by trypan blue dye exclusion using a Bio-Rad automatic cell counter (Bio-Rad, Berkeley, CA, USA). The number of NB4 cells was calculated in triplicate after 24 or 48 h of culture.Apoptotic cells were detected with an Alexa Fluor 488 annexin V/Dead cell apoptosis kit (Invitrogen, Carlsbad, CA, USA) by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). Early apoptotic cells were defined as Annexin-V-positive/propidium iodide- (PI-) negative cells. The experiments were repeated 3 times.For analysis of the cell cycle distribution, NB4 cells were washed 3 times with cold phosphate-buffered saline (PBS), fixed with 70% ethanol, and incubated at −20°C for more than 12 h. Before examination, the cells were washed with cold PBS and stained with 0.5 mL PI staining buffer (200 mg/mL RNase A and 50 μg/mL PI in PBS). The mixture was incubated at 37°C for 30 min in the dark, and cell cycle distribution was analyzed by flow cytometry (BD Biosciences). The experiments were repeated 3 times.


## 2.3. miRNA and mRNA Extraction and Real-Time Quantitative Polymerase Chain Reaction (RTq-PCR)

miRNA was extracted with a mirVana miRNA isolation kit (AM1560, Applied Biosystems, Foster City, CA, USA). MaturemiR-146a was detected by TaqMan miRNA assay as previously reported [13], using a TaqMan_MicroRNA Reverse Transcription Kit (4366597, Applied Biosystems) and PCR 9700 sequence detection system (Applied Biosystems). U48 was used as an internal control. miRNA levels were expressed as relative 2
-
Δ
CT values.mRNAs of other genes (Smad4,PML/RARα,ABL, andGAPDH), excluding miRNAs, were extracted using a QIAampRNA blood mini kit (Qiagen, Valencia, CA, USA), and 10 μg mRNA was used in each reverse transcription reaction, carried out with Superscript II reverse transcriptase and random primers (Invitrogen). Gene expression was determined by RTq-PCR as previously described [14] on a PCR 9700 sequence detection system.ABL andGAPDH were used as internal controls. mRNA levels were expressed as relative 2
-
Δ
CT values.


## 2.4. Western Blot Analysis

A total of 5 × 106 cells of each experimental group were harvested and subsequently were washed twice with phosphate-buffered saline (PBS). Cells were then lysed with lysis buffer (300 mM NaCl, 0.5% NP-40, 1 mM DTT, 200 mM PMSF, protease inhibitor tablet) for 20 minutes. The protein concentration of the cell lysates was quantified using a bicinchoninic acid (BCA) protein Assay Kit (Kangchen, China). 20 μg protein extracted from each experimental sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes and blotted with rabbit polyclonal anti-Smad4 (1 : 5000, Cell Signaling Technology, Danvers, MA, USA) or rabbit monoclonal anti-GAPDH (1 : 2000, Cell Signaling Technology) antibodies. Protein bands were visualized with the use of enhanced chemiluminescence reagent (Pierce), according to the manufacturer’s instructions, and band intensities were analyzed using Bandscan 5.0 (Glyko, Hayward, CA, USA).


## 2.5. Patients, Sample Collection, and Therapeutic Methods

Thirty-two APL patients and ten iron deficiency anemia patients as control who attended Renji Hospital were enrolled in this study. The clinical characteristics of these patients are shown in Table1. Bone marrow was collected from patients at diagnosis or after therapy. APL patients were treated with ATRA (30 mg/m2) and daunorubicin (DNR; 60 mg/m2)/idarubicin (IDA; 8 mg/m2) daily for 3 days. Arsenic trioxide was used at 10 mg daily until patients achieved complete remission. The details of these patients including treatment protocol can be found in Table 2. Protein extracts from 14 patients were used for western blotting, including 6 pairs of samples taken before and after therapy. Written informed consent for participation in this study was obtained from all patients. The study was approved by the Ethics Committee of Renji Hospital (number 81270626).Table 1
Clinical characteristics of APL patients.
Characteristic
Mean ± SD
Age
42.22 ± 2.82
Gender (male/female)
13/19
WBC count (109/L)
12.22 ± 3.45
BM blast cell percentage (%)
84.70 ± 1.89
PML/RARα
419.81 ± 48.26
Th/Ts ratio
1.48 ± 0.10Table 2
Clinical variables for controls and APL patients.
Patients ID
Gender
Age
WBC (×109/L)
PML/RARa : ABL relative ratio
Th/Ts
Smad4/GAPDH
Therapy
Control
C1
M
44
6.32
NA
0.98
NA
NA
C2
F
34
4.67
NA
1.03
NA
NA
C3
F
59
5.96
NA
0.61
NA
NA
C4
F
28
11.30
NA
1.31
NA
NA
C5
M
24
8.35
NA
1.87
NA
NA
C6
F
63
5.90
NA
1.55
NA
NA
C7
F
30
7.40
NA
1.07
NA
NA
C8
M
38
3.76
NA
1.12
NA
NA
C9
F
68
7.75
NA
2.05
NA
NA
C10
M
35
4.81
NA
2.11
NA
NA
APL
A1
F
53
0.80
174
2.03
1.4061
ATRA + ATO + DNR
A2
F
56
3.20
285
1.69
1.2696
ATRA + ATO + IDA
A3
F
67
22.40
713.5
1.01
0.7683
ATRA + ATO + DNR
A4
M
33
6.56
493.5
1.49
0.9575
ATRA + ATO + IDA
A5
M
76
57.44
829.5
0.57
0.3519
ATRA + DNR
A6
F
33
7.50
537
1.39
NA
ATRA + DNR
A7
M
64
1.50
169
1.85
1.7688
ATRA + ATO + IDA
A8
F
44
2.30
416.5
1.88
1.3570
ATRA + ATO + IDA
A9
F
34
3.80
434
1.72
NA
ATRA + ATO + IDA
A10
F
63
6.30
501.5
0.97
1.1272
ATRA + ATO + IDA
A11
M
28
64.60
1048
0.36
0.2464
ATRA
A12
F
24
1.05
225.5
1.95
NA
ATRA + ATO + IDA
A13
F
24
1.60
143
1.8
2.0794
ATRA + ATO + IDA
A14
M
30
7.60
221
1.61
0.8966
ATRA + ATO + IDA
A15
F
38
34.10
617
0.49
NA
ATRA + DNR
A16
M
21
2.30
378
1.15
NA
ATRA + ATO + DNR
A17
F
23
0.70
183
2
1.5293
ATRA + DNR
A18
M
48
2.60
331
1.27
NA
ATRA + DNR
A19
F
24
3.90
411
1.86
NA
ATRA + ATO + IDA
A20
F
34
9.70
612
1.31
NA
ATRA + ATO + DNR
A21
F
60
1.30
173
1.87
1.9294
ATRA + ATO + IDA
A22
F
50
1.60
199
1.91
NA
ATRA + ATO + IDA
A23
M
30
1.60
189
1.85
NA
ATRA + ATO + IDA
A24
F
56
1.10
191
1.99
NA
ATRA + ATO + IDA
A25
M
51
3.20
367
1.7
NA
ATRA + ATO + IDA
A26
F
24
2.20
336
1.91
NA
ATRA + ATO + IDA
A27
M
40
1.00
156
1.97
NA
ATRA + ATO + IDA
A28
M
15
28.30
729
0.93
NA
ATRA + ATO + IDA
A29
F
62
0.60
201
2.13
NA
ATRA + ATO + DNR
A30
M
50
41.40
854
0.64
NA
ATRA + ATO + IDA
A31
F
44
67.20
1137
0.3
0.3373
ATRA + ATO + IDA
A32
M
52
1.50
179
1.82
NA
ATRA + ATO + IDA
M: male; F: female; WBC: white blood cell; ATRA:all  trans retinoic acid; ATO: arsenic trioxide; DNR: daunorubicin; IDA: idarubicin; NA: not available.


## 2.6. Statistical Analyses

To investigate whethermiR-146a expression correlated with clinical quantitative variables (e.g., WBC counts), we used Pearson correlation analysis within SPSS 16.0 (SPSS Inc., Chicago, IL, USA). The Mann-Whitney U test was carried out to assess the differential expression ofmiR-146a for statistical significance. Data from western blotting were analyzed by paired t-tests using SAS version 12.6. All results were presented as the mean ± standard deviation (SD). P values of less than 0.05 were considered statistically significant.


## 3. Results

### 3.1. Ectopic Expression ofmiR-146a Affected Cell Apoptosis and Proliferation but Did Not Affect the Cell Cycle in NB4 Cells Lines

To examine the functional role ofmiR-146a in APL cells, we transfectedmiR-146a into NB4 cells and collected the cells 48 h after transfection. The results showed that forced expression ofmiR-146a significantly inhibited apoptosis and increased cell viability in NB4 cells, while knockdown ofmiR-146a expression using an anti-miR-146a inhibitor increased apoptosis and reduced cell viability significantly (Figures 1(a) and 1(b)). Overexpression ofmiR-146a in NB4 cells did not affect the cell cycle distribution (Figure 1(c)). The expression ofmiR-146a before and after transfection was confirmed by RTq-PCR (data not shown). Thus,miR-146a may function as an oncogene in leukemogenesis.The functional role ofmiR-146a. (a) Forced expression ofmiR-146a by transfection of cells with pre-miR-146a with lipidosome significantly inhibited apoptosis, whereas downregulation ofmiR-146a by anti-pre-miRmiR-146a inhibitor significantly increased apoptosis in NB4 cells. (b) Forced expression ofmiR-146a significantly increased, whereas downregulation ofmiR-146a significantly decreased cell proliferation in NB4 cells. (c) The cell cycle was not affected after ectopic expression ofmiR-146a. Normalized mean values of 3 independent experiments and standard errors (means ± SDs) are shown. *, P
≤
0.05; *
*, P
≤
0.001 (paired t test).
(a)
(b)
(c)


### 3.2. Exogenous Expression ofmiR-146a Affected Endogenous Smad4 Protein Expression in APL Cells

As shown in our previous research, Smad4 protein expression was repressed by more than 30% inmiR-146a-transfected 293T cells [13]. We overexpressedmiR-146a to confirm the effects ofmiR-146a on Smad4 protein expression in NB4 cells. The results showed thatmiR-146a significantly reduced Smad4 protein levels but did not affectsmad4 mRNA expression, as compared with the scramble control group. In contrast, transfection withmiR-146a inhibitor increased Smad4 protein (Figures 2(a) and 2(b)), indicating thatmiR-146a regulated the expression of Smad4 protein in NB4 cells. To confirm the relation in primary leukemia cells, the expression levels ofmiR-146a and Smad4 were examined in 14 APL samples. The results showed that Smad4 expression was inversely correlated withmiR-146a expression in APL samples (Figure 2(c)).ExogenousmiR-146a and endogenous Smad4 expression. (a) Western blot analysis of Smad4 protein expression in NB4 cells after transfection withmiR-146a duplex,miR-146a inhibitor duplex, or scrambled duplex. (b) RTq-PCR analysis ofSmad4 gene expression in NB4 cells after transfection withmiR-146a duplex,miR-146a inhibitor duplex, or scrambled duplex. (c)miR-146a was inversely correlated with Smad4 protein expression in samples from 14 APL patients. (d)miR-146a and Smad4 protein expression analysis in samples from 6 APL patients at diagnosis and after complete remission using RTq-PCR and western blotting. Normalized mean values of 3 independent experiments and standard errors (means ± SDs) are shown. *, P
≤
0.05; *
*, P
≤
0.001 (paired t test).
(a)
(b)
(c)
(d)As we previously demonstrated in NB4 cells, ATRA could suppressmiR-146a expression, which subsequently increased the expression of Smad4 protein [13]. To study the effects of ATRA onmiR-146a and Smad4 expression levels in primary cells, 6 pairs of samples from APL patients before and after ATRA treatment were analyzed. The results showed thatmiR-146a expression decreased, while Smad4 protein levels increased after treatment with ATRA, as compared to matched samples before therapy (Figure 2(d)).These data demonstrated that the negative correlation betweenmiR-146a and Smad4 protein level may be an indicator of disease progression and treatment outcomes.


### 3.3.miR-146a Expression Was Associated with the Clinical Characteristics of Patients

Next, we investigated the correlations betweenmiR-146a expression and patient characteristics, including age, WBC count, blast cell percentage in the bone marrow or peripheral blood, PML/RARα expression levels, and Th/Ts ratios. Compared to the controls, we found averagemiR-146a expression levels were higher in APL patients (Figure 3(a)). We found a positive correlation betweenmiR-146a expression and peripheral WBC counts (Figure 3(b)). However, no correlations were found betweenmiR-146a expression and age or blast percentage in peripheral or bone marrow (data not shown).miR-146a expression and clinical characteristics of 32 patients. (a)miR-146a levels in APL patients, controls, and NB4 cells. (b)miR-146a levels were positively correlated with white blood cell (WBC) counts. (c)miR-146a was positively correlated with PML/RARα gene expression. (d)miR-146a expression was negatively correlated with the Th/Ts ratio.
(a)
(b)
(c)
(d)To investigate the correlations betweenmiR-146a and PML/RARα and Th/Ts, 32 samples from patients with APL were examined. The results showed that patients with highermiR-146a expression exhibited higher levels of PML/RARα (Figure 3(c)), but lower Th/Ts ratios (Figure 3(d)).


## 3.1. Ectopic Expression ofmiR-146a Affected Cell Apoptosis and Proliferation but Did Not Affect the Cell Cycle in NB4 Cells Lines

To examine the functional role ofmiR-146a in APL cells, we transfectedmiR-146a into NB4 cells and collected the cells 48 h after transfection. The results showed that forced expression ofmiR-146a significantly inhibited apoptosis and increased cell viability in NB4 cells, while knockdown ofmiR-146a expression using an anti-miR-146a inhibitor increased apoptosis and reduced cell viability significantly (Figures 1(a) and 1(b)). Overexpression ofmiR-146a in NB4 cells did not affect the cell cycle distribution (Figure 1(c)). The expression ofmiR-146a before and after transfection was confirmed by RTq-PCR (data not shown). Thus,miR-146a may function as an oncogene in leukemogenesis.The functional role ofmiR-146a. (a) Forced expression ofmiR-146a by transfection of cells with pre-miR-146a with lipidosome significantly inhibited apoptosis, whereas downregulation ofmiR-146a by anti-pre-miRmiR-146a inhibitor significantly increased apoptosis in NB4 cells. (b) Forced expression ofmiR-146a significantly increased, whereas downregulation ofmiR-146a significantly decreased cell proliferation in NB4 cells. (c) The cell cycle was not affected after ectopic expression ofmiR-146a. Normalized mean values of 3 independent experiments and standard errors (means ± SDs) are shown. *, P
≤
0.05; *
*, P
≤
0.001 (paired t test).
(a)
(b)
(c)


## 3.2. Exogenous Expression ofmiR-146a Affected Endogenous Smad4 Protein Expression in APL Cells

As shown in our previous research, Smad4 protein expression was repressed by more than 30% inmiR-146a-transfected 293T cells [13]. We overexpressedmiR-146a to confirm the effects ofmiR-146a on Smad4 protein expression in NB4 cells. The results showed thatmiR-146a significantly reduced Smad4 protein levels but did not affectsmad4 mRNA expression, as compared with the scramble control group. In contrast, transfection withmiR-146a inhibitor increased Smad4 protein (Figures 2(a) and 2(b)), indicating thatmiR-146a regulated the expression of Smad4 protein in NB4 cells. To confirm the relation in primary leukemia cells, the expression levels ofmiR-146a and Smad4 were examined in 14 APL samples. The results showed that Smad4 expression was inversely correlated withmiR-146a expression in APL samples (Figure 2(c)).ExogenousmiR-146a and endogenous Smad4 expression. (a) Western blot analysis of Smad4 protein expression in NB4 cells after transfection withmiR-146a duplex,miR-146a inhibitor duplex, or scrambled duplex. (b) RTq-PCR analysis ofSmad4 gene expression in NB4 cells after transfection withmiR-146a duplex,miR-146a inhibitor duplex, or scrambled duplex. (c)miR-146a was inversely correlated with Smad4 protein expression in samples from 14 APL patients. (d)miR-146a and Smad4 protein expression analysis in samples from 6 APL patients at diagnosis and after complete remission using RTq-PCR and western blotting. Normalized mean values of 3 independent experiments and standard errors (means ± SDs) are shown. *, P
≤
0.05; *
*, P
≤
0.001 (paired t test).
(a)
(b)
(c)
(d)As we previously demonstrated in NB4 cells, ATRA could suppressmiR-146a expression, which subsequently increased the expression of Smad4 protein [13]. To study the effects of ATRA onmiR-146a and Smad4 expression levels in primary cells, 6 pairs of samples from APL patients before and after ATRA treatment were analyzed. The results showed thatmiR-146a expression decreased, while Smad4 protein levels increased after treatment with ATRA, as compared to matched samples before therapy (Figure 2(d)).These data demonstrated that the negative correlation betweenmiR-146a and Smad4 protein level may be an indicator of disease progression and treatment outcomes.


## 3.3.miR-146a Expression Was Associated with the Clinical Characteristics of Patients

Next, we investigated the correlations betweenmiR-146a expression and patient characteristics, including age, WBC count, blast cell percentage in the bone marrow or peripheral blood, PML/RARα expression levels, and Th/Ts ratios. Compared to the controls, we found averagemiR-146a expression levels were higher in APL patients (Figure 3(a)). We found a positive correlation betweenmiR-146a expression and peripheral WBC counts (Figure 3(b)). However, no correlations were found betweenmiR-146a expression and age or blast percentage in peripheral or bone marrow (data not shown).miR-146a expression and clinical characteristics of 32 patients. (a)miR-146a levels in APL patients, controls, and NB4 cells. (b)miR-146a levels were positively correlated with white blood cell (WBC) counts. (c)miR-146a was positively correlated with PML/RARα gene expression. (d)miR-146a expression was negatively correlated with the Th/Ts ratio.
(a)
(b)
(c)
(d)To investigate the correlations betweenmiR-146a and PML/RARα and Th/Ts, 32 samples from patients with APL were examined. The results showed that patients with highermiR-146a expression exhibited higher levels of PML/RARα (Figure 3(c)), but lower Th/Ts ratios (Figure 3(d)).


## 4. Discussion

Recent studies have mainly focused on analyzing the function ofmiR-146a in hematopoietic cell differentiation; these data have indicated thatmiR-146a expression is finely tuned during cell differentiation andmiR-146a-mediated expression of target genes plays an important role in this process [15]. Moreover, one study demonstrated that overexpression ofmiR-146a in mouse hematopoietic stem/progenitor cells results in a transient myeloid expansion in vivo [16].miR-146a and its predicted target gene Smad4 were identified in a previous study using luciferase assays [13]. In our previous study,miR-146a expression was decreased during retinoid acid induction in NB4 cells, accompanied by upregulation of Smad4 protein. These results suggested thatmiR-146a may play specific roles in APL genesis or ATRA-induced cell differentiation. Elucidating the role ofmiR-146a will improve our understanding of the malignant progression of APL.In our present study, transfection withmiR-146a mimics increased proliferation, while inhibition ofmiR-146a expression resulted in decreased cell growth and increased apoptosis in NB4 cells. However, ectopic expression ofmiR-146a had no effect on cell cycle in NB4 cells. These findings are consistent with several previous studies. Starczynowski et al. investigated the role ofmiR-146a in hematopoiesis by using retroviral infection and overexpression ofmiR-146a in mouse hematopoietic stem/progenitor cells, followed by bone marrow transplantations. The transplantation of these cells resulted in a transient myeloid expansion [16]. Moreover,miR-146a has been found to play an antiapoptotic role during T cell activation [17].Some other results were inconsistent with our results. OverexpressionmiR-146a in hepatic stellate cell (HSC) inhibited cell proliferation by enhancing cell apoptosis [18]. Treating HL60 cells with demethylating agents increasedmiR-146a expression, and forced expression ofmiR-146a decreases cell proliferation in HL60 cells [19]. Chen et al. also showed thatmiR-146a was upregulated during phorbol 12-myristate 13-acetate (TPA)-induced differentiation of HL-60 cells [20].miR-146a has also been shown to be important in the development and maintenance of cancers, including anaplastic thyroid cancer, pancreatic carcinoma, gastric cancer, breast cancer, prostate cancer, glioma, and cervical cancer [21–28]. All of these data showmiR-146a expression, function, and regulation in cancers or hematopoietic malignancies may vary in terms of the types of tissues or cells affected. Additionally,miR-146a expression in different cancers or tissues is thought to be regulated primarily by point mutations, histone deacetylation, or promoter methylation [29].The transforming growth factor (TGF)-β signaling pathway and its downstream target proteins, including Smads, are known to repress cell proliferation in leukemia cells. Inactivation of the TGF-β/Smad signaling pathway can induce the development of leukemia in humans. Smad4 is the only Co-Smad that has been identified in mammals and is known to play a key role in the TGF-β1/Smad signaling cascade [30]. Furthermore, mutations in Smad4 can block nuclear translocation of this gene, which is associated with the pathogenesis of acute myelogenous leukemia [31]. Our data showed that transient transfection withmiR-146a mimics suppressed Smad4 protein expression in NB4 cells, while transfection with anmiR-146a inhibitor increased Smad4 protein expression in these cells. Like other miRNAs,miR-146a can modulate cellular activities, such as cell proliferation, differentiation, and migration, by targeting mRNAs encoding specific proteins. Overexpression ofmiR-146a in a model of osteoarthritis (OA) was accompanied by downregulation of Smad4 in vivo [32]. In this OA model study,miR-146a was proposed to contribute to OA pathogenesis by impairing the TGF-β signaling pathway through targeted inhibition of Smad4 in cartilage [32]. Interestingly,miR-146a overexpression in HSC can also reduce Smad4 protein levels without affecting its mRNA expression [18]. These discoveries have provided insights contributing to our understanding of the mechanisms involved inmiR-146a/Smad4 signaling transduction.miR-146a expression is significantly downregulated during monocytic differentiation in NB4 cells in response to chemical inducers vitamin D and phorbol 12-myristate 13-acetate (PMA) [33]. Our previous study showed thatmiR-146a is downregulated during NB4 differentiation induced by ATRA [13]. In the current study,miR-146a expression was decreased, accompanied by increased Smad4 protein expression in APL patients exhibiting complete remission. These data suggested thatmiR-146a expression was correlated with APL cell differentiation.In our analysis,miR-146a was found to be positively correlated with WBC counts in patients, which is consistent with our in vitro data. In another study,miR-146a was overexpressed in hematopoietic stem cells, and the modified cells were then transplanted into recipients’ bone marrow. Transplantation of these cells resulted in a transient myeloid expansion [16]. This result also showed thatmiR-146a indeed affected cell proliferation. PML/RARα complexes can bind to the predicted PML/RARα binding site in the promoter region ofmiR-146a, and the repressive effects of PML/RARα complexes can be blocked by ATRA, even in primary blast cells [11]. In our study, expression of the PML/RARα fusion gene was decreased after patients achieved complete remission following retinoid acid induction, andmiR-146a expression decreased simultaneously, indicating thatmiR-146a was regulated by PML/RARα in the leukemogenesis of APL.miR-146a is abundantly expressed in regulatory T cells and T helper type-1 cells as compared with naïve mature T cells and T helper type-2 [34].miR-146a contributes to the regulation of the type I interferon signal transduction pathway, which is important for immune surveillance [17, 21]. Our data showed a negative correlation betweenmiR-146a and the Th/Ts ratio in APL patients (Figure 3(c)), suggesting that overexpression ofmiR-146a may impair immune surveillance in APL patients.In conclusion, our data demonstrated thatmiR-146a played an important role in APL cells by increasing cell proliferation and inhibiting cell apoptosis, at least in part via the tumor-suppressor Smad4.miR-146a expression was associated with peripheral WBC counts, Th/Ts ratios, and PML/RARα expression levels in APL patients, suggesting thatmiR-146a may represent a novel biomarker for APL and could be used to evaluate the effectiveness of chemotherapy for APL.



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*Source: 150604-2014-08-05.xml*