Metformin alters the expression profiles of microRNAs in human pancreatic cancer cells

diabetes research and clinical practice 96 (2012) 187–195

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Diabetes Research and Clinical Practice journ al h ome pa ge : www .elsevier.co m/lo cate/diabres

Metformin alters the expression profiles of microRNAs in human pancreatic cancer cells Weiguang Li, Yaozong Yuan *, Liya Huang, Minmin Qiao, Yongping Zhang Department of Gastroenterology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, 197 Ruijin Second Road, Shanghai 200025, PR China

article info

abstract

Article history:

Aims: To investigate the effect of metformin on the expression profiles of microRNAs in

Received 27 July 2011

human pancreatic cancer cells.

Accepted 19 December 2011

Methods: MicroRNAs real-time PCR Array was applied to investigate differentially expressed

Published on line 14 January 2012

miRNAs in Sw1990 cells treated with or without metformin. Stem-loop real time RT-PCR was

Keywords:

26a on cell growth, apoptosis, invasion and migration abilities were respectively examined

used to confirm the results of the array assay in Sw1990 and Panc-1 cells. The effects of miRMetformin

by CCK8 assay, Apoptosis assay, Matrigel invasion and migration assay. HMGA1 was proved

MicroRNAs

to be a target of miR-26a by Luciferase reporter assay, Real-time PCR and Western-blotting.

Pancreatic cancer

Results: Nine miRNAs were significantly up-regulated in metformin treated cells. Metformin

HMGA1

up-regulated the expression of miR-26a, miR-192 and let-7c in a dose-dependent manner. Forced expression of miR-26a significantly inhibited cell proliferation, invasion, migration and increased cell apoptosis, whereas knockdown of miR-26a obtained the opposite effect. Furthermore, we demonstrated that HMGA1, an oncogene, is a direct target of miR-26a. Nude mice xenograft models confirmed that metformin up-regulated the level of miR-26a and surpressed the expression of HMGA1 in vivo. Conclusion: These observations suggested that modulation of miRNA expression may be an important mechanism underlying the biological effects of metformin. # 2011 Elsevier Ireland Ltd. All rights reserved.

1.

Introduction

Pancreatic carcinoma (PaC) is the fourth leading cause of cancer-related deaths in Western countries and has the poorest survival rate (<5%) among the common malignancies [1,2]. Type 2 diabetes mellitus (T2DM), obesity, cigarette smoking and family history of PaC have been recognized as risk factors for PaC [3]. Recently, antidiabetic therapies have been shown to affect the risk of PaC. a epidemiologic report linked the administration of metformin with a 62% reduced risk of pancreatic cancer in patients with type 2 diabetes mellitus [4].

Metformin (1,1-dimethylbiguanide hydrochloride) is an oral anti-hyperglycemic medication used in the management of T2DM that functions primarily through improved insulin sensitivity and decreased hepatic gluconeogenesis [5]. Epidemiologic studies showed that diabetic patients treated with metformin had a lower incidence of cancer of any type, as well as cancer-related motality, when compared with patients on other treatments [6,7]. In addition, Metformin were proved to prevent carcinogen-induced pancreatic cancer induction in hamsters maintained on high-fat diets [8] and inhibit the growth of breast and colon cancer cells [9,10]. However, the precise mechanisms involved remain incompletely understood. So far, several potential mechanisms for anti-neoplastic

* Corresponding author. Tel.: +86 13901872276; fax: +86 021 64333548. E-mail addresses: [email protected], [email protected] (Y. Yuan). 0168-8227/$ – see front matter # 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2011.12.028

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action of metformin have been suggested, including AMPkinase pathway (AMPK) activation [11,12], p53 activation [10], downregulation of cyclin D1 [12,13] and suppression of HER2 oncoprotein expression [11]. MicroRNAs (miRNAs) are a conserved class of non-coding 20–22 nt small RNAs that regulate gene and protein expression by binding to mRNA leading to mRNA degradation or inhibition of translation [14,15]. Genome-wide studies have demonstrated that miRNA genes are frequently located at cancer-associated genomic regions or in fragile sites, indicating the potential roles of miRNAs in tumorigenesis [16]. Aberrant miRNA expression has also been frequently reported in PaC [17]. However, very few compounds, not to mention metformin, which affect cell growth and/or development, have been shown to affect miRNA expression. In this present study, we elucidated the miRNAs signature in response to metformin treatment in human pancreatic cancer cells. Our results indicated that metformin alters specific miRNA expression in human pancreatic cells, including miR-26a. The effect of miR-26a on apoptosis, proliferation, migration, and invasion ability of pancreatic cancer cells were further investigated. Moreover, we identified HMGA1, an oncogene, as a target of miR-26a. It therefore appears that miR-26a-related changes are important effects of metformin.

2.

Materials and methods

2.1.

Cell lines and cultures

Pancreatic ductal cancer cell lines Sw1990 and Panc-1 were conserved in our own laboratory and were cultured in DMEM medium (GIBCO) with 10% fetal bovine serum (FBS; GIBCO) in a humidified incubator at 37 8C with an atmosphere of 5% CO2.

2.2.

Mice xenografts and tissue samples

About 2  106 Sw1990 cells were harvested and implanted into the right flanks of female nu/nu mice. The animals were randomized into control and treated groups (6 mice per group). Treatment was initiated when the tumors reached a mean diameter of 2 mm, and the first day of treatment in both cases was designated as day 0. For injection, metformin was dissolved in sterile saline and was given once daily i.p. at 250 mg/kg (100 mL/mouse). The control group received vehicle only (100 mL saline). Tumor volume (V) was measured with an external caliper every 4 d and it was calculated as V = 0.52 (length  width2). The treatment was continued until any of the tumors reached 1.5 cm in length, when all the animals were sacrificed and the tumors removed for further investigation.

2.3.

MicroRNAs real time PCR array

Sw1990 cells (3  105) were plated on 6-well plates in DMEM with 10% FBS. After 24 h of incubation at 37 8C, the cells were treated with or without 5 mmol/L metformin. The cultures were incubated for 2 d, then the total RNA was isolated from cell samples using Trizol reagent (Invitrogen) following the manufacturer’s protocol. Then, cDNA synthesis was

performed using Universal cDNA synthesis kit (Exiqon). The expression levels of 372 human mature microRNAs were examined using the miRCURY LNATM Universal RT microRNA PCR system, Ready-to-use human panel I (Exiqon, kangchen, China). Briefly, total RNA containing microRNA was polyadenylated, and cDNA was synthesized using a poly(T) primer with a 30 degenerate anchor and a 50 universal tag. Then, cDNA was served as a template for microRNA quantitative real-time PCR (qPCR) using miRCURY LNA Universal RT microRNA PCR kit (Exiqon). The miRNA Ready-to-use human panel I is a 384-well PCR plate containing dried down LNATM primer sets for one real-time PCR reaction per well. Three small RNA (U6snRNA, SNORD38B, SNORD49A) and three microRNA (miR-103, miR191 and miR-423-5p) reference genes are included on the panel. The amplification profile was denatured at 95 8C for 10 min, followed by 40 cycles of 95 8C for 10 s and 60 8C for 60 s. At the end of the PCR cycles, melting curve analyses were performed. All reactions were done in triplicate. Expression levels of mature mRNAs were evaluated using comparative CT method (2DCT).

2.4.

Stem-loop real-time reverse transcription (RT)-PCR

The miRNAs (miR-26a, miR-192 and let-7c) were quantitated by stem-loop real time RT-PCR to confirm the reliability of the miRNA array assay. In brief, Sw1990 and Panc-1 cells (3  105) were seeded on 6-well plates in DMEM with 10% FBS. After 24 h of incubation, the cells were treated with different dose of metformin (0–10 mmol/L). Total RNA was isolated 48 h later. 0.2–0.5 mg of total RNA was reverse transcribed to cDNA using target-specific stem-loop primers (Supplementary Materials). cDNA in water was added 5 mL of the 2 SYBR green master mix (Applied Biosystems Inc., Foster City, USA), 400 nmol/L of gene-specific primer and water to 10 mL. The reactions were amplified at 95 8C for 10 min followed by 40 cycles at 95 8C for 15 s and 60 8C for 60 s. U6 small nuclear RNA (U6) served as the endogenous control. The relative amount of each miRNA to U6 was described using the formula 2DCt where DCt = (Ct miRNA  CtU6). Each sample was run in triplicate.

2.5.

Oligonucleotide construction and cell transfection

After metformin treatment, the expression of miR-26a was increased most obviously in Sw1990 cells and Panc-1 cells, so we further investigated the functional roles of miR-26a in pancreatic cancer cells. MiR-26a mimics, miR-26a inhibitor and negative control siRNA oligonucleotides were chemosynthesized (Shanghai GenePhama Co. Ltd.). The oligonucleotides used in these studies were has-miR-26a mimics: 50 -UUCAAGUAAUCCAGand 50 -CCUAUCCUGGAUUACUUGAAUU-30 . GAUAGGCU-30 Mimics negative control: 50 -UUCUCCGAACGUGUCACGUTT-30 and 50 -ACGUGACACGUUCGGAGAATT-30 , hsa-miR-26a inhibitor: 50 -AGCCUAUCCUGGAUUACUUGAA-30 . MicroRNA inhibitor negative control: 50 -CAGUACUUUUGUGUAGUACAA-30 . Cells were cultured to 80–90% confluence after being seeded into 6-well plates and were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. For transient transfection, cells in each well of a 6-well plate were transfected with 12.5 ml

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miRNA inhibitor or 7.5 ul miRNA mimic oligonucleotides. Transfection efficiency was evaluated by GFP expression in control vector or real-time PCR.

2.6.

Apoptosis assay

At 72 h after transfection, apoptosis was detected using Annexin V-FITC Apoptosis Detection Kit (Biovision, USA). Results were calculated by the percentage of apoptotic cells in all cells counted.

2.7.

Matrigel invasion assay

At 48 h after transfection, the invasive ability of the cells was assayed using Transwells (8 mm pore size, Corning Costar Corp.). The Transwells were put into the 24-well plates. First, 0.1 ml Matrigel (50 mg/ml, BD Biosciences) was added onto the plate surface and incubated for 2 h, and then the supernatant was removed. Freshly trypsinized and washed Panc-1 or Sw1990 cells were suspended in DMEM containing 1% fetal bovine serum. Then 0.1 ml of the cell suspension (1  105 cells) was added to the upper chamber of each insert that was coated with Matrigel. Next, 0.6 ml of DMEM containing 10% fetal bovine serum was added into the lower compartment, and the cells were allowed to invade for 24 h at 37 8C in a 5% CO2 humidified incubator. After incubation, the cells were fixed with 95% absolute alcohol and followed by crystal violet stain. The number of migrated cells on the lower surface of the membrane was counted under a microscope in 10 fields with magnification of 200. Each experiment was performed in triplicate.

2.8.

Cell migration

At 48 h after transfection, the ability of Panc-1 or Sw1990 cells to migrate was detected using Transwells [8 mm pore size, Corning Costar Corp.]. The Transwells were put into the 24well plates. Freshly trypsinized and washed cells were suspended in DMEM containing 1% fetal bovine serum. 5  104 cells/well were placed in the top chamber of each insert (BD Biosciences, NJ), with the non-coated membrane. 0.6 ml of DMEM containing 10% fetal bovine serum was added into the lower chambers. After incubating for 24 h at 37 8C in a 5%CO2 humidified incubator, After incubation, the cells were fixed with 95% absolute alcohol and followed by crystal violet stain. The number of migrated cells on the lower surface of the membrane was counted under a microscope in 10 fields with magnification of 200. Each experiment was performed in triplicate.

2.9.

Cell proliferation assay

A total of 104 Panc-1 or Sw1990 cells per well were plated in 96-well plates before transfection and cultured for 24 h in normal conditions. They were then transfected with hsamiR-26a mimics or hsa-miR-26a inhibitor along with paired negative controls. The cells were incubated at 37 8C for 48 h. Cell proliferation was assessed using Cell Counting Kit 8 (Dojindo, Tokyo, Japan) according to manufacturer’s protocol.

2.10.

189

Real-time PCR for HMGA1

Total RNA (1 mg) isolated from tissue or cell samples (48 h after transfection) was reverse transcribed using reverse transcription kit (Promega, Madison, WI, USA). Quantitative real-time PCR was performed with a SYBR Green-real-time PCR master mix kit (Toyobo, Tokyo, Japan) for detection of HMGA1. GAPDH was used as endogenous control (i.e., reference gene). Forward (F) and reverse (R) primer sequences were as follows: HMGA1 (F) 50 -ACTGGAGTCTCCTGTGGTGTGT-30 , HMGA1 (R) 50 -AGTGCTATTTCCCCTCCCTTC-30 ; GAPDH (F) 50 -ACGGATTTG-GTCG TATTGGGC-30 , and GAPDH (R) 50 -TTGACGGTGCCATGGAATTTG-30 . PCR was performed for 15 s at 95 8C and1 min at 60 8C for 40 cycles.

2.11.

Western blotting

Western blot was performed according to standard procedures. Total protein was isolated from tumor samples or pancreatic cancer cells (48 h after transfection). The concentration was measured by BCA protein assay kit (Pierce, USA). To detect the activation of HMGA1, ten micrograms of each sample was resolved using 6% SDS-PAGE and transferred onto Immobilon-P membranes (Millipore). The bands were incubated with 10% nonfat dry milk in Tris-buffered saline– 0.1% Tween-20 to block the nonspecific binding sites and incubated with a primary antibody: goat polyclonal antihuman HMGA1 (diluted 1:500; Abcam) overnight at 4 8C. After rewarming and repeated washing three times, 10 min for each one, the membranes were incubated with horseradishperoxidase-conjugated anti-goat secondary antibody (Santa Cruz Biotechnology), diluted 1:2000 for 1 h at room temperature. The signal intensity of the bands was determined by Image J software.

2.12.

Luciferase reporter assay

To investigate whether HMGA1 expression was regulated by miR-26a, a dual-luciferase reporter assay was performed. A fragment of 30 UTR of HMGA1 containing miR-26a binding site was amplified by PCR. Amplification of HMGA1 used the following primers: Forward: 50 -GAATTCGCCAAATGTTCATCCTCA-30 , Reverse: 50 -CATATGTGTCACCCATCCTACC TGC-30 . As a negative control, the mutated binding site of the 30 UTR sequence was amplified using the primers: Forward: 50 TTTTTTATAGTTCATACTGCAAATAGGAAACCA-30 ;Reverse:50 GCAGTATGAACTATAAAAAAAATATCCTTTTC-30 . Products were cloned into the luciferase reporter construct pGL3-miReport vector. All constructs were verified by sequencing. The wild-type or a mutated 30 UTR of HMGA1 and miR26a mimics were co-transfected into HEK-293T cells. After 48 h of incubation, firefly and Renilla luciferase activity was measured with the dual luciferase reporter assay system (Promega).

2.13.

Statistical analysis

Data are expressed as the mean  sd unless otherwise noted. The differences between groups were analyzed using

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a two-tailed Student’s t-test when only two groups were present and the null hypothesis was rejected at the 0.05 level.

3.

Results

3.1. Metformin treatment alters miRNA expression profiles To study the responses of miRNAs to metformin, miRNA real time PCR array analysis of miRNA expression was conducted with total RNAs extracted from SW1990 pancreatic cells treated with or without 5 mmol/L metformin. Differential expression between metformin-treated and non-treated cells was defined using a cut off value of 2-fold change. we observed that 9 miRNAs were significantly upregulated (P < 0.05; 2.86fold on average) in metformin treated cells when compared with non-treated cells (Fig. 1A).

3.2. Confirmatory studies with differentially expressed miRNAs by stem-loop real-time PCR MiR-26a, miR-192, and let-7c showed 4.2-fold, 3.6-fold and 3fold higher expression in Sw1990 cells treated with 5 mmol/L metformin compared with non-treated cells, respectively. To validate that these three miRNAs were overexpressed in

metformin treated pancreatic cancer cells, stem-loop realtime quantitative analysis was done in Sw1990 and Panc1 cells treated with different doses of metformin (0–10 mmol/ L). Metformin up-regulated the expression of miR-26a, miR192 and let-7c in a dose-dependent manner in Sw1990 and Panc-1 cells (Fig. 1B and C). The results obtained by stem-loop quantitative real-time PCRs were comparable with and confirmed the miRNA real-time PCR array data.

3.3. miR-26a Inhibits Cell Proliferation and Increases Cell Apoptosis We investigated the potential tumor suppressive activity of miR-26a in Sw1990 and Panc-1 cells. First, we tested miR-26a expression using stem-loop real-time PCR. It showed an increase or decrease after transfected with miR-26a mimics or anti-miR-26a inhibitor. We observed a significant decrease (Sw1990: 1.51  0.13 vs 2.58  0.15; Panc-1: 1.69  0.10 vs 2.88  0.10; P < 0.01) in proliferation after transfection of miR-26a mimics. In contrast, anti-miR-26a inhibitor significantly (Sw1990: 2.82  0.09 vs 2.04  0.11; Panc-1: 2.65  0.10 vs 2.10  0.16; P < 0.01) increased cell proliferation. These data indicate that cell proliferation can be significantly suppressed by increase of miR-26a expression. We further investigated the effect of miR-26a on apoptosis and found that apoptosis was increased dramatically (Fig. 2;

Fig. 1 – Effect of metformin on miRNAs expression in Sw1990 cell line. (A) Nine miRNAs showed significant increase in levels of expression after 5 mmol/L metformin treatment for 48 h compared with non-treated cells. The cut off line for miRNA expression change was 2-fold. (B and C) Sw1990 and Panc-1 cells were incubated with increasing concentrations of metformin (0.5–10 mmol/L). Then the expression of the three miRNAs were detected by Stem-loop real-time reverse transcription (RT)-PCR. Metformin up-regulated the expression of miR-26a, miR-192 and let-7c in a dose-dependent manner. Data are presented as means W sd.

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Fig. 2 – Enhancement of miR-26a increases apoptosis in Sw1990 and Panc-1 cells. The proportion of apoptosis cells induced by transfection of miR-26a mimics is significantly greater. (**P < 0.01)than that induced by transfection of mismatched sequence miRNA negative control. Data are shown as means W sd.

Sw1990: 43.30  4.76% vs 12.84  1.43%; Panc-1: 42.10  2.93% vs 15.29  0.75; P < 0.01) in Sw1990 and Panc-1 cells 72 h after transfection with miR-26a mimics, suggesting that miR-26a may function as a strong apoptosis promoter in human pancreatic cancer cells.

3.4. miR-26a regulates pancreatic cancer cell invasion and migration in vitro In cell invasion and migration assay, we observed that depletion of miR-26a significantly enhanced the ability of Sw1990 cells to migrate and invade through the matrigelcoated membranes or the non-matrigel-coated membranes towards serum-containing medium (Fig. 3A; P < 0.01). Increased expression of miR-26a significantly suppressed the ability of Sw1990 cells to migrate and invade through matrigel-coated membranes or non-matrigel-coated membranes towards serum-containing medium (Fig. 3B; P < 0.01), when compared with the control cells. Similar results were found in Panc-1 cells (Fig. 3C and D; P < 0.01). These results indicate that miR-26a may be important in the progression of pancreatic cancer through inhibiting cell invasion and migration.

3.5.

HMGA1 is a target of miR-26a

We then searched for the potential targets of miR-26a using currently available major prediction programs, including TargetScan [18], MiRanda [19] and PicTar [20]. We found that

HMGA1, an oncogene, was predicted as a target of miR-26a. HMGA1 can be regulated negatively by miR-26a at both transcriptional level (Fig. 4A; P < 0.05) and protein level (Fig. 4B) in Sw1990 and Panc-1 cells. Furthermore, we performed a luciferase reporter assay and observed a significant decrease (Fig. 4D; P < 0.01) in luciferase activity in the presence of pGL3-miR-26a-HMGA1 in HEK-293T cell compared the controls. To validate whether HMGA1 is a direct target of miR-26a, we mutated the miR-26a binding site in the 30 UTR of HMGA1 (Fig. 4C) and observed loss of repression. These results all indicate HMGA1 is a direct target of miR-26a.

3.6. Metformin inhibits the growth of Sw1990 xenografts, upregulates miR-26a and suppresses the expression of HMGA1 in vivo Administration of metformin strikingly decreased the growth of Sw1990 cells xenografted in nude mice. At the end of the experiment (day 24), the tumor volumes of Sw1990 xenografts were 340.24  42.43 mm3 in the control group and 166.81  23.55 mm3 in the metformin-treated group (Fig. 5A, P < 0.01). We further investigated the expression of miR-26a and HMGA1 in these tumors. The expression of miR-26a were significantly higher in metformin-treated group, compared with control group (Fig. 5B, P < 0.01). HMGA1 was downregulated at both transcriptional level (Fig. 5C; P < 0.01) and protein level (Fig. 5D) in metformin-treated group, compared with control group. These results indicate metformin regulate the expression of miR-26a and HMGA1 in vivo.

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Fig. 3 – Effect of miR-26a on tumor cell migration, invasion of Sw1990 cells and Panc-1 cells. (A) Invasion and migration assay. Representative fields of invasion (down) or migration (up) cells on the membrane (left). (magnification of 200T). Average invasion or migration cell number per field (right). The invasion or migration cell number of Sw1990 transfected with miR-26a-inhibitors is drastically increased than that transfected with pairing negative control. **P < 0.01, n = 10. (B) The invasion or migration cell number of Sw1990 Cells transfected with miR-26a-mimics is dramatically decreased than that transfected with pairing negative control. **P < 0.01, n = 10. (C and D) Similar results were found in Panc-1 cells. **P < 0.01, n = 10.

4.

Discussion

It is clear from recent studies that there is a close correlation between miRNAs and human malignancy [21,22]. miRNAs can act as both oncogenes and/or tumor suppressor genes within the molecular architecture of gene regulatory net-works, thereby contributing to the development of cancer. For

example, miR-155 can promote tumor invasion and metastasis in breast cancer by downregulating its target, RhoA [23]. MiR-217 was found to inhibit pancreatic cancer cell growth through targeting KRAS [24]. MiR-218 inhibits the invasion and metastasis of gastric cancer by targeting the Robo1 receptor [25]. Therefore, miRNAs may provide useful diagnostic and prognostic markers for cancer diagnosis or treatment and serve as potential therapeutic targets or tools.

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Fig. 4 – HMGA1 is target of miR-26a. (A) The expression of HMGA1 was analyzed by qRT-PCR. HMGA1 was decreased in Sw1990 or Panc-1 cells transfected with miR-26a mimics compared to cells transfected with pairing negative control, while HMGA1 was increased in Sw1990 or Panc-1 cells transfected with anti-miR-26a inhibitor compared to cells transfected with pairing negative control (B) HMGA1 expression level in Sw1990 or Panc-1 cells after transfection with anti-miR-26a inhibitor or miR-26a mimics detected by western blotting. (C) Predicted consequential pairing of target 30 -UTR region of HMGA1 (wild type or mutated) and miR-26a mature sequence. (D) Luciferase activity on the presence of both wild-type HMGA1 30 UTR or mutant and miR-26a (i.e., 30 UTR+miR-26a) were compared with those of the controls (i.e., 30 UTR+vacant vector and 30 UTR+miR-181a. (*P < 0.05; **P < 0.01).

The biguanide metformin is the most widely prescribed drug for the treatment of type 2 diabetes, worldwide. Some studies showed that metformin has potent growth-inhibitory and proapoptotic effects on pancreatic cancer in vitro and in vivo [4,26]. The goal of our present study was to determine whether metformin alters the expression profiles of miRNAs in human pancreatic cancer cells. Our miRNAs real time PCR array experiments showed that metformin up-regulated the expression of 9 miRNAs (such as miR-26a). Stem-loop realtime PCR experiments indicated that metformin up-regulate the expression of miR-26a, miR-192 and let-7c in a dosedependent manner, confirming the expression patterns we found in our miRNAs real time PCR array experiments. We then focused our attention on miR-26a. MiR-26a was found to remarkably inhibit cell proliferation and increase apoptosis in Sw1990 and Panc-1 cells. In addition, we showed that knockdown of miR-26a could dramatically increase cell invasion and migration; on the contrary, overexpression of miR-26a could lead the opposite effect. Thus, our data suggest that miR-26a may be important in the development of pancreatic cancer.

In addition, we identified an oncogene, HMGA1, as a target of miR-26a. The high-mobility group A (HMGA) proteins could bind the minor groove of AT-rich DNA regions, through their amino-terminal DNA-binding domain, which consists of three short basic repeats, the so-called AT-hooks [27]. HMGAs are involved in several cellular processes such as gene and miRNA expression, chromatin and nucleosome remodeling, DNA replication, apoptosis and DNA repair [28–30]. HMGAs also has a causal role in cellular transformation [28]. The overexpression of HMGAs is commonly found in almost all human malignant neoplasias, representing a poor prognostic marker correlated with the presence of metastasis, reduced survival and resistance to anti-cancer therapies [31]. Our data suggested that HMGA1 expression has a negative correlation with the expression of miR-26a in pancreatic cancer cell lines at transcriptional level and protein level. Applying specific miR-26a oligonucleotides can decrease HMGA1 expression, negatively regulated pancreatic cancer cell metastasis. The current study showed that the expression of HMGA1 was significantly decreased in Sw1990 xenografts treated with metformin. Taken these results together, we

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Fig. 5 – Metformin inhibits the growth of Sw1990 xenografts and regulates the expression of miR-26a and HMGA1 in vivo. (A) Administration of metformin (250 mg/kg. i.p. daily) strikingly decreased the growth of Sw1990 cells xenografted in nude mice. At the end of the experiment, all animals were sacrificed and the tumors were removed. The tumors were much more heavier in control group than that in metformin-treated group. (B) The relative expression of miR-26a detected by qPCR. Data were normalized by U6 RNA. The expression of miR-26a were strikingly increased in metformin treated group. (C) The relative expression of HMGA1 detected by qPCR. Data were normalized by GAPDH. The expression of HMGA1 were significantly decreased in metformin treated group. (D) The expression of HMGA1 detected by Western blotting. Four tumors were chosen randomly from each group for Western blotting. The expression of HMGA1 were significantly higher in control group when compared with metformin-treated group. Data are shown as means W sd (*P < 0.05; **P < 0.01). M, metformin; C, control.

hypothesize that miR-26a-HMGA1 is one of the important effects of metformin on pancreatic cancer cells. In conclusion, we used miRNAs real time PCR array technology to analyze the expression of 372 miRNAs following treatment with metformin in Sw1990 cells. Three metforminresponsive miRNAs, miR-26a, miR-192, and let-7c, showed

major and consistent effects, which were validated by stemloop real-time PCR in Sw1990 and Panc-1 cells. Furthermore, manipulating the expression of miR-26a altered the expression of its target genes, HMGA1. This suggests an important and novel new mechanism by which metformin mediates its potent effects on cell growth and apoptosis.

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Authors contributions [11]

Weiguang Li and Yaozong Yuan designed the research; Weiguang Li performed the majority of experiments; Liya Huang, Minmin Qiao and Yongping Zhang were involved in editing the manuscript; Weiguang Li and Yaozong Yuan analyzed the data and wrote the paper.

Conflict of interest

[12]

[13]

The authors declare that they have no conflict of interest. [14]

Acknowledgments We gratefully acknowledge the assistance of Qi Wang and Dandan Song in the Department of Gastroenterology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine for their kind help.

[15] [16] [17]

[18]

Appendix A. Supplementary data [19]

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.diabres.2011. 12.028.

[20] [21]

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