Ann Surg Oncol DOI 10.1245/s10434-014-4255-8

ORIGINAL ARTICLE – TRANSLATIONAL RESEARCH AND BIOMARKERS

MicroRNA-29b is a Novel Prognostic Marker in Colorectal Cancer Akira Inoue, MD1, Hirofumi Yamamoto, MD, PhD1, Mamoru Uemura, MD, PhD1, Junichi Nishimura, MD, PhD1, Taishi Hata, MD, PhD1, Ichiro Takemasa, MD, PhD1, Masakazu Ikenaga, MD, PhD2, Masataka Ikeda, MD, PhD3, Kohei Murata, MD, PhD4, Tsunekazu Mizushima, MD, PhD1, Yuichiro Doki, MD, PhD1, and Masaki Mori, MD, PhD1 1

Department of Surgery, Gastroenterological Surgery, Graduate School of Medicine, Osaka University, Osaka, Japan; Department of Surgery, Osaka Rosai Hospital, Osaka, Japan; 3Department of Surgery, National Hospital Organization, Osaka National Hospital, Osaka, Japan; 4Department of Surgery, Suita Municipal Hospital, Osaka, Japan

2

ABSTRACT Purpose. Recent studies have suggested that microRNA29 (miR-29) family members may play important roles in human cancer by regulating cell proliferation, differentiation, apoptosis, migration, and invasion. The present study aimed to investigate the clinical significance and biological function of miR-29b in colorectal cancer (CRC). Methods. Real-time polymerase chain reaction was used to quantify miR-29b expression. The association between miR-29b and survival was evaluated in 245 patients with CRC. We transfected an miR-29b mimetic into CRC cells to explore the functional role of miR-29b in vitro, based on a proliferation assay, flow cytometry, and Western blotting. Results. In clinical samples of CRC, miR-29b expression was significantly reduced in tumor tissues compared with normal mucosa (p \ 0.012). Multivariate survival analyses indicated that miR-29b expression was an independent prognostic factor for disease-free survival (p = 0.026), lymph node metastasis (p = 0.004), and pathological T classification (p = 0.002). In a multivariate analysis of 5year overall survival, we found a similar association between lymph node metastasis, pathological T classification, venous invasion, and miR-29b expression (p = 0.013). In vitro, low

Electronic supplementary material The online version of this article (doi:10.1245/s10434-014-4255-8) contains supplementary material, which is available to authorized users. Ó Society of Surgical Oncology 2014 First Received: 20 April 2014 H. Yamamoto, MD, PhD e-mail: [email protected]

Ki-67-positive staining showed that administration of the mimic-miR-29b reduced proliferation of CRC cells. An Annexin V apoptosis assay and flow cytometric analysis revealed that miR-29b induced apoptosis and arrested the cell cycle at the G1/S transition. Moreover, miR-29b inhibited the expression of MCL1 and CDK6. Conclusions. Our findings indicated that miR-29b may be a useful, novel, prognostic marker and may play important roles in regulating apoptosis and cell cycle in CRC

INTRODUCTION MicroRNAs (miRNAs) are small (*22 nucleotide), endogenous, non-coding RNAs that regulate gene expression at the post-transcriptional level through RNA interference.1,2 Accumulating evidence indicates that miRNAs are aberrantly expressed in human cancers and contribute to tumorigenesis. Consequently, miRNAs provide diagnostic and prognostic information. In addition, miRNAs have been shown to alter cancer phenotypes by targeting biological pathways critical to tumorigenesis.3,4 For example, altered expression of miR-21, miR-31, miR-34a, miR143, and miR-145 were closely related to the clinicopathologic features and prognoses of colorectal cancer (CRC).5–7 Circulating miR-378 is a reliable, hemolysis-independent biomarker for CRC.8 Moreover, miR-30b is a tumor suppressor in human CRC that targets KRAS, PIK3CD, and BCL2.9 However, it remains largely unknown how miRNAs regulate target gene and signaling pathways associated with CRC tumorigenesis. Recent studies have suggested that microRNA-29 (miR29) family members may play important roles in various

A. Inoue et al.

types of human malignancies by regulating cell proliferation, differentiation, apoptosis, migration, and invasion. Mott et al. showed that miR-29b was an endogenous regulator of myeloid cell leukemia sequence 1 (MCL1), and induced apoptosis in cholangiocarcinoma cell lines.10 Li et al. demonstrated that miR-29b suppressed cell proliferation and induced apoptosis via regulation of the BCR/ABL1 protein in chronic myelogenous leukemia.11 Zhao et al. identified miR-29 as a prognostic marker and pathogenic factor that targeted cyclin-dependent kinase 6 (CDK6) in mantle cell lymphoma.12 Moreover, miR-29b was identified as a promising candidate for miRNA-based therapeutics for lung cancer and acute myeloid leukemia because it regulated DNA methylation (i.e. DNMT1, DNMT3A, and DNMT3B), cell-cycle progression (i.e. CDK6), and apoptosis (i.e. MCL1).13–15 However, to our knowledge no studies have shown the role of miR-29b in CRC. In this study, we hypothesized that miR-29b might regulate progression of CRC through the target molecules and might serve as a useful biomarker to estimate prognosis of patients with CRC.

hsa-miR-29b: ID 000413, and RNU6B: ID 001093. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed with the 7900HT Sequence Detection System (Applied Biosystems). Amplification data were normalized to RNU6B expression, and relative expression was quantified using the 2-DDCt method. Messenger RNA Expression Cellular mRNA expression levels were measured with the LightCycler TaqManÒ Master (Roche Diagnostics, Basel, Switzerland). First, 1,000 ng total RNA was reverse transcribed with the Reverse Transcription System (Promega, Inc., Madison, WI, USA), and the resulting cDNA was then amplified with the specific primers listed in the Appendix. The qRT-PCR reactions were performed with the LightCyclerÒ 2.0 System (Roche Diagnostics) according to the manufacturer’s protocol. Amplification data were normalized to ACTB (actin, beta) expression. Cell Lines and Cell Culture

MATERIALS AND METHODS Patients and Clinical Samples We obtained primary CRC tissues from 245 patients who underwent primary tumor resections between 2003 and 2005 at Osaka University Hospital and two related hospitals. Tumor samples were immersed in RNAlaterÒ (Ambion Inc., Austin, TX, USA) and stored at -80 °C until RNA extraction. The patients exhibited CRC disease stage I (N = 34), stage II (N = 63), stage III (N = 104), and stage IV (N = 44). The mean follow-up period was 4.5 years. All patients provided written informed consent, in accordance with guidelines approved by the Institutional Review Board of Ethics at each institute. RNA Isolation Total RNA, including miRNA, was isolated from tissue samples and cell lines with an miRNeasy kit (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer’s protocol. Total RNA concentration and purity were reassessed with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, DE, USA). Micro RNA (miRNA) Expression We measured miRNA levels in tissues and cells with the TaqMan miRNA Assay (Applied Biosystems). First, 5 ng RNA was reverse transcribed; the resulting complementary DNA (cDNA) was amplified with TaqMan miRNA assays;

Human CRC cell lines DLD1, HT29, HCT116, SW480, and CaCO2 were obtained from the American Type Culture Collection (Rockville, MD, USA) in 2001. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in a humidified atmosphere of 95 % air and 5 % carbon dioxide. Transient Transfection of miRNA Cells were transfected with 30 nmol/L (final concentration) of the mimic-miR precursor miRNA molecule, hsamiR-29b (Gene Design Inc., Osaka, Japan; sequence: 50 UAGCACCAUUUGAAAUCAGUGUU-30 ), with LipofectamineÒ RNAiMAX (Invitrogen, Darmstadt, Germany), in 6- or 24-well plates. The mimic-miR negative control (Gene Design Inc.; sequence: 50 -UAAAUGUACUGCGC GUGGAGAGGAA-30 ) was used as a control. Cell Proliferation Cells were seeded at a density of 5–7 9 104 cells/well in 24-well dishes, and cultured for 72 h to determine proliferation. Cell counting was performed with an automatic hematology analyzer, Celltac (Nihon Kohden, Tokyo, Japan). Ki-67 Index of Cell Proliferation Cells were cultured in six-well plates until confluence. After transfection and 72 h of growth, cells were fixed

Prognostic Value of miR-29b in CRC

with 4 % paraformaldehyde, and incubated in blocking buffer (1 % BSA, 0.1 % Triton-X-100, 0.002 % Tween-20 in 1 % TBS) for 1 h. The fluorescent antibody, anti-Ki-67 (1:400, Dako, Glostrup, Denmark), was added to cells and incubated overnight at 4 °C. A secondary antibody (1:1000, Alexa Fluor 546 goat anti-mouse IgM, Invitrogen) was then added for 1 h. Cells were mounted with ProLongÒ Gold Antifade Reagent, and stained with DAPI (Life Technologies Japan, Tokyo, Japan). Images were captured with a BZ-9000 fluorescence microscope (KEYENCE, Osaka, Japan). Apoptosis Apoptosis was assayed in control and treated cells with an Annexin V assay kit (Invitrogen). Briefly, 2 9 105 cells were harvested at various intervals after treatment and re-suspended in 200 lL of binding buffer. Annexin V-fluorescein isothiocyanate (FITC) and 1 lg/mL propidium iodide (PI) were added, and cells were incubated for 15 min in a dark environment. The reaction was stopped by adding 300 lL of 19 binding buffer, and labeled cells were analyzed by flow cytometry with a FACSAria II (BD Bioscience, San Jose, CA, USA). Cell-Cycle Analysis The cell cycle was assessed by flow cytometry. First, 60 h before analysis, cells were seeded in six-well plates and starved with serum-free medium to synchronize the cell cycle. Forty-eight hours before analysis, cells were transfected with mimic-miR-29b or the negative control miR. The cultures were transferred into medium containing 10 % fetal bovine serum, and at various intervals; Hoechst 33342 solution (1 mg/ml stock) [DOJINDO, Kumamoto, Japan] was added at 1.68 ll volume per well. After incubating for 30 min, cells were harvested and analyzed with a FACSAria II flow cytometer (BD Bioscience). Western Blot Analysis To isolate the proteins, cells were collected from sixwell plates, and lysed in lysis buffer. The proteins in each sample were resolved with sodium dodecyl sulfate– polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes, and incubated with monoclonal antibodies against MCL1 (1:1000, #4572, Cell Signaling Technology, Inc., Danvers, MA, USA), CDK6 (1:500, #3136), or ACTB (1:4000, SigmaAldrich). After incubating with secondary antibodies, signals were detected with the ECL Detection System (GE Healthcare, Little Chalfont, UK).

Statistical Analysis Statistical analyses were performed with the JMP10 program (SAS Institute, Cary, NC, USA) and GraphPad Prism version 6.00 for Windows (GraphPad Software, San Diego, CA, USA). Clinicopathologic factors were compared using the v2 test, and continuous variables were compared using the Student’s t test. Survival curves were computed using the Kaplan–Meier method. After univariate analysis of the potential predictive factors affecting disease-free survival (DFS) and overall survival (OS), only significant variables were used in the multivariate analysis, using the Cox proportional hazard model and the log-rank test to calculate hazard ratios (HR) and 95 % confidence intervals (CI). In vitro analysis data are expressed as the mean ± standard deviation, and p-values \ 0.05 were considered statistically significant. RESULTS MiR-29b Expression in Normal Colonic Mucosa and Colorectal Cancer (CRC) Tissues The expression of miR-29b was detected in 42 CRC tissue samples and adjacent normal mucosa tissue samples (electronic supplementary Fig. S1). CRC tissues showed significantly lower miR-29b expression compared with normal mucosa tissues (p = 0.012; Fig. 1a). Effect of MiR-29b Expression on Prognosis of Patients with CRC We analyzed miR-29b expression in tissue samples from 245 patients with CRC. Patients were divided into two groups: those under the median (low) and those above the median (high) of miR-29b expression. Kaplan–Meier survival analysis revealed that high miR-29b expression was significantly associated with higher 5-year DFS and higher 5-year OS (p = 0.03 and 0.02, respectively; Fig. 1b, c). In sub-analyses by each stage, we found that miR-29b expression had a prognostic impact on 5-year DFS solely in patients with stage III CRC (p = 0.0003; electronic supplementary Fig. S2). A clinicopathologic survey (Table 1) indicated no significant correlation between miR-29b expression and the disease characteristics. Table 2 shows the results of univariate and multivariate analyses of clinicopathologic factors related to DFS. Univariate analysis indicated that lymph node metastasis (p = 0.0003), pathological T classification (p = 0.0001), and low miR-29b expression (p = 0.034) were significantly related to the 5-year DFS. Multivariate analysis indicated that low miR-29b expression was an independent predictor of a reduced 5-year DFS, with a

A. Inoue et al.

a 60 Expression level

FIG. 1 Expression of miR-29b in CRC tissue samples. a CRC tissues (n = 42) showed significantly lower miR-29b expression compared with normal tissues (n = 42). b, c Kaplan–Meier survival analysis revealed that high miR-29b expression (DFS, n = 101; OS, n = 122) was significantly associated with higher 5-year DFS rates (p = 0.03; stages I– III, median follow-up 1,656 days) and higher 5-year OS rates (p = 0.02; stages I–IV, median follow-up; 1,657 days) compared with low miR-29b expression (DFS, n = 100; OS, n = 123). The cut-off line between high and low expression was set at the median miR-29b expression level in each cohort. miR-29 microRNA-29, CRC colorectal cancer, DFS disease-free survival, OS overall survival

P = 0.012

40

20

0 Normal Mucosa (n=42)

b

%

5-year Disease Free Survival

Colorectal Tumor (n=42)

c

5-year OverallSurvival

%

High miR-29b expression (n=101)

High miR-29b expression (n=122)

100

100

80

80

Low miR-29b expression (n=100)

60 40

60

Low miR-29b expression (n=123)

40

P = 0.02

P = 0.03 20

20

0

0 0

500

1000

1500

Days after surgery

relative risk (RR) of 2.165, and a 95 % CI of 1.093–4.511 (p = 0.026). In addition, low miR-29b was predictive of lymph node metastasis (p = 0.004) and a pathological T classification (p = 0.002) [Table 2]. Similarly, multivariate analysis showed that the 5-year OS was associated with lymph node metastasis (p = 0.009), a pathological T classification (p = 0.027), venous invasion (p = 0.035), and miR-29b expression (p = 0.013) [electronic supplementary Table S1]. MiR-29b Inhibited Proliferation and Induced Apoptosis in CRC Cells In Vitro In DLD1, HT29, HCT116, and SW480 cells, mimicmiR-29b treatment significantly inhibited cell growth compared with parental cells (p \ 0.01) or negative control miR-treated cultures at 72 h (p \ 0.01 for each; Fig. 2a and electronic supplementary Fig. S3a). We next examined the effect of miR-29b on apoptosis of CRC cells. Mimic-miR-29b was transfected into CRC cells and analyzed every 24 h, up to 72 h, with flow cytometry. DLD1, HT29, HCT116, and SW480 cells transfected with mimic-miR-29b showed significantly more apoptosis compared with those transfected with the negative control miR (p \ 0.05 for each; Fig. 2b and electronic supplementary Fig. S3b).

2000

2500

0

500

1000

1500

2000

2500

Days after surgery

Transfection of miR-29b and Cell-Cycle Arrest in CRC Cells In Vitro We further explored the role of miR-29b in CRC cell proliferation by examining the distribution of cells in different stages of the cell cycle. At 24 h, DLD1, HT29, HCT116, and SW480 cells transfected with mimic-miR29b showed a significantly larger percentage of cells in the G1/G0 phase and a smaller percentage of cells in the G2/M phase compared with those transfected with the negative control miR (p \ 0.05 for each; Fig. 3 and electronic supplementary Fig. S4). Moreover, after 48 h, the expression of Ki-67, a well-known marker of proliferation, was dramatically decreased in DLD1 and HT29 cells transfected with mimic-miR-29b compared with those transfected with the negative control miR (electronic supplementary Fig. S5). MiR-29b Inhibited Myeloid Cell Leukemia Sequence 1 (MCL1) and Cyclin-Dependent Kinase 6 (CDK6) Expression in CRC Cells At 48 h after transfection, miR-29b expression was significantly increased in each cell transfected with mimicmiR-29b compared with cells transfected with negative control miR and untransfected (parent) cells (p \ 0.01;

Prognostic Value of miR-29b in CRC TABLE 1 miR-29b expression and clinicopathologic factors Clinicopathologic variables

High miR-29b expression (n = 122)

Low miR-29b expression (n = 123)

p value

75

78

NS

47 4.90 ± 2.04

45 4.64 ± 1.97

NS

Rectum

51

59

NS

Colon

71

64

Sex Male Female Tumor size (cm, mean ± SD) Location

Histological grade Well–Mod

114

113

Others

8

10

NS

Pathological T stage T1, T2

25

16

T3, T4

97

107

UICC classification 7th edition I 20

NS

14

II

28

35

III

57

47

IV

17

27

?

75

69

-

47

54

?

65

66

-

57

57

NS

Lymphatic invasion NS

Venous invasion NS

Lymph node metastasis ?

69

66

-

53

57

NS

miR-29 microRNA-29, NS not significant, Well–Mod well to moderately differentiated adenocarcinoma, SD standard deviation, UICC Union for International Cancer Control

electronic supplementary Figs. S6a and S7a). At 48 h after transfection, MCL1 mRNA was significantly decreased in HT29, HCT116, and CaCO2 cells transfected with mimicmiR-29b compared with cells transfected with the negative control miR (p \ 0.05 for each; Fig. 4a and electronic supplementary Figs. S6b and S7b). However, DLD1 and SW480 cells showed no significant effect of mimic-miR29b on MCL1 mRNA expression (Fig. 4a and electronic supplementary Fig. S6b). CDK6 mRNA was significantly decreased in DLD1, HT29, HCT116, and SW480 cells transfected with mimic-miR-29b compared with cells transfected with negative control miR (p \ 0.05 for each; Fig. 4b and electronic supplementary Fig. S6c). At 48 h after transfection, Western blots showed that MCL1 protein expression decreased to some extent in DLD1, HT29, HCT116, and CaCO2, and CDK6 protein expression was dramatically downregulated in DLD1 and HT29 cells transfected with mimic-miR-29b compared with controls (Fig. 4c and electronic supplementary Figs. S6d and S7c). DISCUSSION The miR-29 family (miR-29a–c) in humans was recently reported to target genes involved in cell proliferation, cell cycle, cell senescence, differentiation, and apoptosis. In human cancers, the miR-29b was shown to effectively regulate tumor occurrence, progression, and metastasis at genetic and epigenetic levels.10,14,16–20 In this present study, we showed that miR-29b expression was significantly decreased in human CRC tissues compared with normal colonic mucosa, and this reduction was significantly correlated to a poor prognosis in patients with CRC. The prognostic impact on disease recurrence was most evident in stage III CRC patients. Transfection of mimicmiR-29b into CRC cells inhibited their growth, induced apoptosis, and arrested cells in the G1 phase. These

TABLE 2 Univariate and multivariate analyses for survival (5-year DFS) Clinicopathologic factors

Univariate

Multivariate

RR

95 % CI

p value

RR

95 % CI

p value

Sex (male/female)

1.105

0.564–2.255

0.774

Tumor size ([3 cm/\3 cm)

1.898

0.804–5.571

0.153

Location (rectum/colon)

1.112

0.565–2.163

0.754

Histological grade (well–mod/others) Pathological T stage (T1, T2/T3, T4)

1.644 7.461

0.395–4.595 2.043–12.427

0.442 0.0001*

5.189

1.029–13.156

0.002*

Venous invasion (±)

1.694

0.871–3.366

0.120

Lymph node metastasis (±)

3.810

1.812–8.984

miR-29b expression (high/low)

2.081

1.054–4.323

0.0003*

2.911

1.378–6.887

0.004*

0.034*

2.165

1.093–4.511

0.026*

DFS disease-free survival, RR relative risk, CI confidence interval, well–mod well to moderately differentiated adenocarcinoma, miR-29 microRNA-29 * p \ 0.05

A. Inoue et al.

a

× 105

DLD1

20

Parent

Cell number

15

*

10

NC

*p < 0.01 Parent NC

5

miR-29b

miR-29b

0 24 h

48 h

× 105

72 h

HT29

15

Parent 10

*

Cell number

NC *p < 0.01

5

Parent NC miR-29b

miR-29b 0 24 h

48 h

72 h

DLD1

b

*p < 0.05

% % of apoptotic cells

NC PI miR29b

50 40

24 h

48 h

*

NC miR-29b

30 20 10 0

Annexin V FITC

24 h

48 h

72 h

72 h

HT29 *p < 0.05

%

NC

% of apoptotic cells

FIG. 2 miR-29b inhibited cell proliferation and induced apoptosis. a DLD1 and HT29 cells transfected with miR-29b showed significantly reduced cell growth compared with untreated (parental) cells or cells treated with the NC miR. Symbols are the mean ± SD of four replicates. Right panels indicate microscopic photographs of each culture at 72 h. Scale bars = 100 lm. The assays were repeated three times and similar results were obtained. b Flow cytometric analysis with Annexin V assays showed that the rate of apoptosis was significantly higher in DLD1 and HT29 cells transfected with mimic-miR29b compared with those transfected with the negative control miR. Right panels indicate the percentage of apoptotic cells expressed as mean ± SD from three different replicates. *p \ 0.05. miR-29 microRNA-29, SD standard deviation, Parent parental cells, NC negative control miRtreated cells, miR-29b miR-29btreated cells

PI miR29b Annexin V FITC 24 h

48 h

72 h

20 15 10

NC miR-29b

* *

*

5 0

24 h

48 h

72 h

Prognostic Value of miR-29b in CRC

DLD1 500 400

Cell numbers

NC

miR29b

G1/G0 = 71.8 % S = 13.1 % G2/M = 15.2 %

500

G1/G0 = 64.9 % S = 20.8 % G2/M = 14.3 %

400 300

300

200

200

200

100

100

100

0 20°C 40°C 60°C 80°C 100°C 120°C 500 400

500

G1/G0 = 69.3 % S = 19.7 % G2/M = 10.9 %

400 300

300

200

200

200

100

100

100

400

DNA content 12 h

Cell numbers miR29b

*

80 60 40

*

0 NC

mR-29b

NC

0h

24 h

mR-29b NC

12 h

mR-29b

24 h

HT29 G1/G0 = 87.1 % S = 7.7 % G2/M = 5.1 %

500

500

G1/G0 = 86.9 % S = 9.4 % G2/M = 3.7 %

400

400

300

300

300

200

200

200

100

100

100

0 20°C 40°C 60°C 80°C 100°C 120°C 500 400

*p < 0.05

20

0 20°C 40°C 60°C 80°C 100°C 120°C

0 20°C 40°C 60°C 80°C 100°C 120°C

0h 500

G1/G0 = 70.3 % S = 17.4 % G2/M = 12.4 %

400

300

G2/M 5 G1/G0 100

0 20°C 40°C 60°C 80°C 100°C 120°C

0 20°C 40°C 60°C 80°C 100°C 120°C 500

G1/G0 = 73.6 % S = 12.8 % G2/M = 13.6 %

G1/G0 = 61.4 % S = 18.6 % G2/M = 19.9 %

400

300

0 20°C 40°C 60°C 80°C 100°C 120°C

NC

500

G1/G0 = 85.7 % S = 7.1 % G2/M = 7.1 %

300

0 20°C 40°C 60°C 80°C 100°C 120°C

G1/G0 = 87.5 % S = 8.6 % G2/M = 3.8 %

0 20°C 40°C 60°C 80°C 100°C 120°C

400

300

300

200

200

200

100

100

100

G2/M 5 G1/G0

*p < 0.05

100

500

500 400

G1/G0 = 50.4 % S = 15.4 % G2/M = 34.2 %

G1/G0 = 57.1 % S = 18.5 % G2/M = 24.3 %

*

80 60 40

*

20

0 20°C 40°C 60°C 80°C 100°C 120°C

0 20°C 40°C 60°C 80°C 100°C 120°C

0 20°C 40°C 60°C 80°C 100°C 120°C

0h

DNA content 12 h

24 h

0 NC

mR-29b

0h

NC

mR-29b NC

12 h

mR-29b

24 h

FIG. 3 miR-29b induced cell-cycle arrest. At 24 h, DLD1 (upper panels) and HT29 (lower panels) cells transfected with miR-29b showed a significantly larger percentage of cells in the G1/G0 phase and a smaller percentage of cells in the G2/M phase compared with

cells treated with NC miR (p \ 0.05 for each difference). Data represent the mean of three different replicates. miR-29 microRNA29, NC negative control

findings are consistent with other previous studies on miR29b in cholangiocarcinoma, myeloid leukemia, mantle cell lymphoma, and lung cancer. Thus, our results suggest that miR-29b may play a crucial role as an anti-miR in CRC. The results also indicate a biological relationship between miR-29b dysregulation and the pathogenesis of human CRC; thus, miR-29b may represent a new therapeutic strategy for CRC treatment and prevention. MCL1, an anti-apoptotic B-cell lymphoma 2 (Bcl-2) family member, is essential for the survival of stem and progenitor cells of multiple lineages.21,22 Studies have shown that MCL1 expression is upregulated in B-cell

lymphomas, chronic myeloid leukemia, multiple myeloma, lung cancer, cholangiocarcinoma, and hepatocellular carcinoma; this upregulation contributes to cancer cell survival, chemoresistance, and disease relapse.10,13,14,16,23,24 Overexpression of MCL1 was demonstrated to be associated with a poor prognostic outcome in several tumors, including myeloma, breast cancer, and leukemia.25 Also, in colon cancer, MCL1 expression was reportedly increased in adenoma and carcinoma tissues compared with normal colonic epithelium.26 Moreover, it was reported that low-dose aspirin and sorafenib co-treatment inhibited proliferation and targeted the anti-apoptotic

A. Inoue et al.

a

Relative MCL1 mRNA expression DLD1

1.5

1.0

1.0

0.5

0.5

0.0

parent

HT29

1.5

*p < 0.05

*

0.0

NC

miR-29b

parent

NC

miR-29b

Relative CDK6 mRNA expression

b

DLD1

*

2.0

HT29

*p < 0.05 1.5

Stephania tetrandra, induced early G1-phase arrest by downregulating several cell-cycle components, including CDK4, CDK6, cyclin D1, p-Rb, and E2F1, in colon cancer cell lines.29 However, no studies examined the relationship between miR-29b expression and MCL1 or CDK6 expression in CRC. Therefore, we investigated the effects of miR29b on MCL1 and CDK6 in CRC in vitro, and found that miR-29b suppressed MCL1 expression and promoted apoptosis in CRC cells. We also found that miR-29b suppressed CDK6 expression, reduced the Ki-67 index, and induced G1 arrest in CRC cells. These findings suggested that miR-29b regulates apoptosis and the cell cycle in colon cancer cells through inhibition of these molecules.

*p < 0.05

1.5

1.0

*

1.0

CONCLUSIONS

0.5 0.5 0.0

parent

0.0 NC

miR-29b

DLD 1

c

parent

NC

miR-29b

HT29

Our findings indicate that miR-29b may be useful as a novel prognostic marker and may play important roles in regulating tumor progression in CRC. Restoration of miR29b expression may represent a potential strategy for miRNA-based therapy against CRC.

MCL1

ACKNOWLEDGMENTS This work was supported by a Grant-in Aid for Scientific Research (KAKENHI) to Hirofumi Yamamoto (numbers 21390360, 30322184, 24390315). The authors are grateful to Masayuki Hiraki, MD, Hidekazu Takahashi, MD, PhD, Xin Wu, Takeshi Kato, MD, PhD, and Junichi Hasegawa, MD, PhD, for their valuable help and discussion in survival analyses.

CDK6 Actin NC miR-29b

NC miR-29b

FIG. 4 miR-29b inhibited MCL1 and CDK6 expression. a (left) No significant change was detected in MCL1 mRNA expression in DLD1 cells transfected with or without mimic-miR-29b. However, (right) MCL1 mRNA expression was significantly reduced in HT29 cells transfected with mimic-miR-29b compared with cells transfected with NC miR and untransfected (parent) cells after 48 h (p \ 0.05). b CDK6 mRNA expression was significantly reduced in DLD1 and HT29 cells transfected with miR-29b compared with cells transfected with NC miR and untransfected cells after 48 h (p \ 0.05). The results are the mean ± SD of triplicates. c Western blot analyses showed that MCL1 protein expression decreased to some extent and CDK6 expression was dramatically downregulated in DLD1 and HT29 cells transfected with mimic-miR-29b compared with controls (NC) after 48 h. Actin bands served as a loading control. miR-29 microRNA-29, mRNA messenger RNA, NC negative control, SD standard deviation

proteins, FLIP (Fas-associated death domain (FADD)-like interleukin-1b converting enzyme (FLICE)-inhibitory protein) and MCL1, in colon cancer cells; these findings suggest that MCL1 is a key target for the prevention and treatment of colon cancer. Aberrations in cell-cycle progression occur in the majority of human carcinomas, including colon cancer. Thus, inhibition of CDKs is a major strategy for tumor prevention and therapy.27,28 Meng et al. reported that tetrandrine, an antitumor alkaloid isolated from the root of

DISCLOSURE Akira Inoue, Hirofumi Yamamoto, Mamoru Uemura, Junichi Nishimura, Taishi Hata, Ichiro Takemasa, Masakazu Ikenaga, Masataka Ikeda, Kohei Murata, Tsunekazu Mizushima, Yuichiro Doki, and Masaki Mori declare no conflicts of interest.

APPENDIX Quantitative PCR primer sequences were: MCL1 (forward primer: 50 -AAGCCAATGGGCAGGTCT-30 ; reverse primer: 50 -TGTCCAGTTTCCGAAGCAT-30 ), CDK6 (forward primer: 50 -TGATCAACTAGGAAAAATCTTGGA-30 ; reverse primer: 50 -GGCAACATCTCTAGGCCAGT-30 ), beta actin [ACTB] (forward primer: 50 -CCAACCGCGAGAAGATGA30 ; reverse primer: 50 -CCAGAGGCGTACAGGGATAG-30 ).

REFERENCES 1. He L, Hannon GJ. MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet. 2004;5:522–31. 2. Chen K, Rajewsky N. The evolution of gene regulation by transcription factors and microRNAs. Nat Rev Genet. 2007;8:93–103. 3. Chen CZ. MicroRNAs as oncogenes and tumor suppressors. N Engl J Med. 2005;353:1768–71. 4. Lu J, Getz G, Miska EA, et al. MicroRNA expression profiles classify human cancers. Nature. 2005;435:834–8.

Prognostic Value of miR-29b in CRC 5. Slaby O, Svoboda M, Michalek J, Vyzula R. MicroRNAs in colorectal cancer: translation of molecular biology into clinical application. Mol Cancer. 2009;8:102. 6. Tazawa H, Tsuchiya N, Izumiya M, Nakagama H. Tumor-suppressive miR-34a induces senescence-like growth arrest through modulation of the E2F pathway in human colon cancer cells. Proc Natl Acad Sci U S A. 2007;104:15472–7. 7. Zhang JX, Song W, Chen ZH, et al. Prognostic and predictive value of a microRNA signature in stage II colon cancer: a microRNA expression analysis. Lancet Oncol. 2013;14:1295– 306. 8. Zanutto S, Pizzamiglio S, Ghilotti M, et al. Circulating miR-378 in plasma: a reliable, haemolysis-independent biomarker for colorectal cancer. Br J Cancer. 2014;110:1001–7. 9. Liao WT, Ye YP, Zhang NJ, et al. MicroRNA-30b functions as a tumour suppressor in human colorectal cancer by targeting KRAS, PIK3CD and BCL2. J Pathol. 2014;232:415–27. 10. Mott JL, Kobayashi S, Bronk SF, Gores GJ. mir-29 regulates Mcl-1 protein expression and apoptosis. Oncogene. 2007;26:6133–40. 11. Li Y, Wang H, Tao K, et al. miR-29b suppresses CML cell proliferation and induces apoptosis via regulation of BCR/ABL1 protein. Exp Cell Res. 2013;319:1094–101. 12. Zhao JJ, Lin J, Lwin T, et al. microRNA expression profile and identification of miR-29 as a prognostic marker and pathogenetic factor by targeting CDK6 in mantle cell lymphoma. Blood. 2010;115:2630–9. 13. Wu Y, Crawford M, Mao Y, et al. Therapeutic delivery of microRNA-29b by cationic lipoplexes for lung cancer. Mol Ther Nucleic Acids. 2013;2:e84. 14. Huang X, Schwind S, Yu B, et al. Targeted delivery of microRNA-29b by transferrin-conjugated anionic lipopolyplex nanoparticles: a novel therapeutic strategy in acute myeloid leukemia. Clin Cancer Res. 2013;19:2355–67. 15. Fabbri M, Garzon R, Cimmino A, et al. MicroRNA-29 family reverts aberrant methylation in lung cancer by targeting DNA methyltransferases 3A and 3B. Proc Natl Acad Sci U S A. 2007;104:15805–10. 16. Garzon R, Heaphy CE, Havelange V, et al. MicroRNA 29b functions in acute myeloid leukemia. Blood. 2009;114:5331–41. 17. Ugalde AP, Ramsay AJ, de la Rosa J, et al. Aging and chronic DNA damage response activate a regulatory pathway involving miR-29 and p53. EMBO J. 2011;30:2219–32.

18. Li Z, Hassan MQ, Jafferji M, et al. Biological functions of miR29b contribute to positive regulation of osteoblast differentiation. J Biol Chem. 2009;284:15676–84. 19. Gebeshuber CA, Zatloukal K, Martinez J. miR-29a suppresses tristetraprolin, which is a regulator of epithelial polarity and metastasis. EMBO Rep. 2009;10:400–5. 20. Schmitt MJ, Margue C, Behrmann I, Kreis S. MiRNA-29: a microRNA family with tumor-suppressing and immune-modulating properties. Curr Mol Med. 2013;13:572–85. 21. Opferman JT, Letai A, Beard C, Sorcinelli MD, Ong CC, Korsmeyer SJ. Development and maintenance of B and T lymphocytes requires antiapoptotic MCL-1. Nature. 2003; 426:671–6. 22. Oltersdorf T, Elmore SW, Shoemaker AR, et al. An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature. 2005;435:677–81. 23. Xiong Y, Fang JH, Yun JP, et al. Effects of microRNA-29 on apoptosis, tumorigenicity, and prognosis of hepatocellular carcinoma. Hepatology. 2010;51:836–45. 24. Chen J, Chen Y, Chen Z. MiR-125a/b regulates the activation of cancer stem cells in paclitaxel-resistant colon cancer. Cancer Invest. 2013;31:17–23. 25. Schwickart M, Huang X, Lill JR, et al. Deubiquitinase USP9X stabilizes MCL1 and promotes tumour cell survival. Nature. 2010;463:103–7. 26. Pennarun B, Kleibeuker JH, Boersma-van Ek W, et al. Targeting FLIP and Mcl-1 using a combination of aspirin and sorafenib sensitizes colon cancer cells to TRAIL. J Pathol. 2013;229:410– 21. 27. Pommier Y, Kohn KW. Cell cycle and checkpoints in oncology: new therapeutic targets [in French]. Med Sci (Paris). 2003; 19:173–86. 28. Malumbres M, Barbacid M. To cycle or not to cycle: a critical decision in cancer. Nat Rev Cancer. 2001;1:222–31. 29. Meng LH, Zhang H, Hayward L, Takemura H, Shao RG, Pommier Y. Tetrandrine induces early G1 arrest in human colon carcinoma cells by down-regulating the activity and inducing the degradation of G1-S-specific cyclin-dependent kinases and by inducing p53 and p21Cip1. Cancer Res. 2004;64:9086–92.