Curcumin inhibits cell growth and invasion through up-regulation of miR-7 in pancreatic cancer cells

Toxicology Letters 231 (2014) 82–91

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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet

Curcumin inhibits cell growth and invasion through up-regulation of miR-7 in pancreatic cancer cells Jia Ma a,1, Binbin Fang b,1, Fanpeng Zeng b,1, Haijie Pang b , Jing Zhang b , Ying Shi a , Xueping Wu c, Long Cheng b , Cong Ma b , Jun Xia a, ** , Zhiwei Wang d, * a

Department of Biochemistry and Molecular Biology, Bengbu Medical College, 2600 Donghai Avenue, Anhui 233030, China Research Center of Clinical Laboratory Science, Bengbu Medical College, Anhui 233030, China Department of Nephrology, The First Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui 233030, China d The Cyrus Tang Hematology Center and Collaborative Innovation Center of Hematology, Jiangsu Institute of Hematology, The First Affiliated Hospital, Soochow University, Suzhou 215123, China b c

H I G H L I G H T S







Curcumin suppressed cell growth, invasion, and induced cell apoptosis. Curcumin increased miR-7 expression and subsequently decreased SET8 expression. Curcumin exerted its anti-tumor activity through regulating miR-7. Targeting miR-7 by curcumin could be a strategy for treatment of pancreatic cancer.

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 June 2014 Received in revised form 16 September 2014 Accepted 19 September 2014 Available online 23 September 2014

Accumulating evidence has revealed that a natural compound curcumin exerts its anti-tumor activity in pancreatic cancer. However, the underlying molecular mechanism remains elusive. Recently, miRNAs have been demonstrated to play a crucial role in tumorigenesis, suggesting that targeting miRNAs could be a promising approach for the treatment of human cancers. In this study, we explored whether curcumin regulates miR-7, leading to the inhibition of cell growth, migration and invasion in pancreatic cancer cells. We observed that curcumin suppressed cell growth, migration and invasion, and induced cell apoptosis, which is associated with increased expression of miR-7 and subsequently decreased expression of SET8, one of the miR-7 targets. These findings demonstrated that targeting miR-7 by curcumin could be a novel strategy for the treatment of pancreatic cancer. ã 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Curcumin miR-7 SET8 Apoptosis Cell growth Pancreatic cancer

1. Introduction Pancreatic cancer (PC) was reported to have 46,420 new cases and 39,590 deaths in the United States in 2014, indicating that PC is one of the most lethal malignancies (Siegel et al., 2014). Although there has been significant progress in the use of

* Corresponding author at: Cyrus Tang Hematology Center, Soochow University, Room 703-3601, 199 Ren Ai Road, Suzhou Industrial Park, Suzhou, Jiangsu 215123, China. Tel.: +86 512 65880899; fax: +86 512 65880929. ** Corresponding author. Tel.: +86 552 3175142; fax: +86 552 3175238. E-mail addresses: [email protected] (J. Xia), [email protected], [email protected] (Z. Wang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.toxlet.2014.09.014 0378-4274/ ã 2014 Elsevier Ireland Ltd. All rights reserved.

diagnostic methods and development of systemic treatments, the overall 5-year survival rate of PC still is less than 6% over the last decades (Bartsch et al., 2012). The high mortality of PC could be due to the inability to detect it at an early stage, lack of effective chemotherapy, and development of drug resistance (Costello et al., 2012). To improve therapeutics of PC, it is important to explore the molecular mechanisms of PC pathogenesis and to develop effective therapeutic agents for the treatment. Curcumin, a phytochemical polyphenol from the rhizomes of Curcuma longa, has been widely studied (Gupta et al., 2013a). As one nutraceutical, curcumin has been shown to play critical roles in anti-inflammatory, antioxidant, wound-healing and antitumor activities (Park et al., 2013). Extensive studies have demonstrated that curcumin is involved in regulating multiple

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Fig. 1. Effect of curcumin on PC cell growth and apoptosis. (A) AsPC-1 and BxPC-3 cells were seeded in 96-well plates at 5000 cells per well and treated with varied concentrations of curcumin for 72 h. After treatment, MTT assay was performed to measure cell growth. (B and C) Apoptotic cell death was measured using Annexin V-FITC/PI method in AsPC-1 cells (B) and BxPC-3 cells (C) without or with curcumin treatment. PI: Propidium iodide; FITC: Fluorescein isothiocyanate; FL1: Fluorescent light channel 1; FL3: Fluorescent light channel 3. UR: upper right, represent late stage apoptotic cells (AnnexinV+ PI +); LR: lower right, represent early apoptotic cells (Annexin+ PI-); UL: upper left, represent necrotic cells (Annexin – PI+); LL: lower left, represent live cells. (D) Confocal scanning laser microscopy was used to measure apoptosis in AsPC-1 and BxPC-3 cells after curcumin treatment.

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cellular signaling pathways including NF-kB (nuclear factor-kB), Akt, Notch, mTOR (mammalian target of rapamycin), and Hedgehog (Beevers et al., 2013; Shehzad and Lee, 2013). More importantly, curcumin has been observed to have safety and nontoxicity at high dose by human clinical trials (Gupta et al., 2013b; Yang et al., 2013). Although accumulating evidence indicated curcumin’s anti-tumor potential, the underlying mechanism has not been fully elucidated. Recently, microRNAs, also known as miRNAs, which belong to small non-coding RNAs, have been intensively studied (van Kouwenhove et al., 2011). It has been known that miRNAs regulate post-transcriptional gene regulation through binding to its targeted mRNA, leading to an increased degradation of the mRNA or a decreased translation rate of the mRNA (Kasinski and Slack, 2011). Several lines of evidence has defined that miRNAs play a crucial role in cancer cell proliferation, apoptosis, migration, invasion and metastasis (Ling et al., 2013). Interestingly, miRNAs could be the product of tumor suppressive or oncogenes, which are dependent on their targets for anti-

tumorigenic or oncogenic activities. There is growing evidence that miRNAs could be governed by natural compounds including curcumin (Sethi et al., 2013). For example, it has been reported that curcumin inhibited invasion and metastasis through regulating miR-21 in colorectal cancer (Mudduluru et al., 2011). Moreover, one study suggests that curcumin inhibited the expression of Bcl-2 via up-regulation of miR-15a and miR-16 in breast cancer (Yang et al., 2010). Studies have revealed that miR-7 was critically involved in the development and progression of human cancers including PC (Chakraborty et al., 2013; Kalinowski et al., 2014). For example, miR-7 inhibited tumor angiogenesis and growth through targeting OGT (O-linked b-Nacetylglucosamine transferase) in murine xenograft glioblastoma (Babae et al., 2014). Furthermore, it has been found that miR7 downregulated XIAP (X-linked inhibitor of apoptosis protein) expression to suppress cell growth and induce apoptosis in cervical cancer cells (Liu et al., 2013). Moreover, miR-7 was recently reported to regulate the stemness, which is controlled by the zinc transporter LIV-1 in PC cells (Unno et al., 2014). Therefore,

Fig. 2. Effect of curcumin on PC cell migration and invasion. (A and B) Cell migration was detected using wound-healing assay in AsPC-1 (A) and BxPC-3 cells (B) after curcumin treatment. (C and D) Cell invasion was measured using Transwell inserts with Matrigel in AsPC-1 (C) and BxPC-3 (D) after curcumin treatment. ***P < 0.05 compared to control.

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in the current study, we explore whether curcumin could regulate miR-7 expression and subsequently control the expression of SET8, one of miR-7 targets, in PC cells. Our results demonstrated that curcumin exerts its anti-tumor activity via up-regulation of miR7 and subsequent down-regulation of SET8 expression in PC cells. 2. Materials and methods 2.1. Cell culture, reagents and antibodies Human AsPC-1 and BxPC-3 PC cells were bought from the American Type Culture Collection (Manassas, VA, USA). AsPC-1 and BxPC-3 cells were cultured in 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin with 5% CO2 in a humidified incubator. Curcumin (CAS number 458-37-7, 99.5% curcumin) and

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MTT [3-(4,5-dimethythiazol- 2-yl)-2,5-diphenyl tetrazolium bromide] were bought from Sigma. Curcumin was dissolved in DMSO to make a 30 mM stock solution and was added directly to the media at different concentrations. Cells were treated with 0.1% DMSO as the control group. The treatment groups with curcumin have less 0.1% DMSO. Antibody against SET8 was purchased from BD Biosciences. Antibody against b-actin and the secondary antibodies were obtained from Santa Cruz Biotechnology. Transwell inserts and Matrigel were purchased from BD Biosciences. 2.2. MTT assay The cell survival study was conducted by MTT assay. The AsPC-1 and BxPC-3 cells (5  103) were seeded in a 96-well culture plate for 24 h and then treated with different

Fig. 3. Curcumin treatment increased miR-7 in PC cells. (A) The expression of miR-7 was detected by real-time RT-PCR in AsPC-1 and BxPC-3 cells after curcumin treatment for 72 h. *P < 0.05 compared to control. (B) Left panel: MTT assay was performed in AsPC-1 after 6 mM curcumin treatment or miR-7 mimics transfection or the combination. Right panel: MTT assay was conducted in BxPC-3 cells after 3 mM curcumin treatment or miR-7 mimics transfection or the combination. *P < 0.05 compared to control; **P < 0.05 compared to curcumin treatment alone or miR-7 mimics alone. (C) MTT assay was performed in AsPC-1 or BxPC-3 cells after curcumin treatment or miR-7 inhibitor transfection or the combination. *P < 0.05 compared to control; **P < 0.05 compared to curcumin treatment alone or miR-7 inhibitor alone.

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concentrations (1–10 mM) of curcumin for 72 h. MTT assay was performed following our previously published procedure (Wu et al., 2013).

2.3. Cell apoptosis assay Cell apoptosis was analyzed using an Annexin V-FITC/PI apoptosis detection kit (Beyotime Institute of Biotechnology,

Fig. 4. Effect of miR-7 mimics on apoptosis and invasion in PC cell. (A and B) Apoptosis was detected by Annexin V-FITC/PI method in AsPC-1 (A) and BxPC-3 (B) cells after curcumin treatment or miR-7 mimics or the combination. (C and D) Cell invasion was measured using Transwell inserts with Matrigel in AsPC-1 (C) and BxPC-3 (D) cells after curcumin treatment or miR-7 mimics or the combination. *P < 0.05 compared to control; **P < 0.05 compared to curcumin treatment alone or miR-7 mimics alone.

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China). The AsPC-1 and BxPC-3 cells were seeded in 6-well plates overnight and then treated with curcumin at indicated concentration for 48 h. The following steps were conducted as the manufacturer’s instruction. The cells were subjected to Annexin V-FITC/PI staining and analyzed using flow cytometer (Yang et al., 2014). 2.4. Wound healing assay The AsPC-1 and BxPC-3 cells (2  105) cells were added to each well of 6-well plates and cultured at 37  C in 5% CO2 until 90–95% confluence. The perpendicular scratch wound was generated by scratching the surface of the plates with a standard 200 ml pipette tip. After wounded monolayers were washed twice to remove the detached cells, the cells were then incubated without or with curcumin for 20 h. The images were captured from each well at 0 h and 20 h.

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were photographed and counted in five randomly selected fields under a microscope. 2.6. MiRNA real-time reverse transcriptase-PCR (RT-PCR) The miRNA RT-PCR was used to detect the alterations of miR-7 expression in PC cells after treatment with curcumin as described previously (Chen et al., 2014). Briefly, 10 ng of total RNA was reversed transcribed into cDNA using TaqMan miRNA hsamiR-7-specific primers (Applied Biosystems). Then real-time PCR was performed using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). RNA U6 was carried out as endogenous control in each sample. The relative expression was analyzed using the comparative Ct method by the 7500 Real-Time System software. 2.7. Levels of mRNAs by real-time reverse transcriptase PCR

2.5. Invasion assay The Transwell assay was performed as described before (Wu et al., 2013). The invasive capacity of AsPC-1 and BxPC-3 cells was performed using Transwell inserts with Matrigel. After incubation for 16 h by curcumin or miR-7 transfection, the non-migrated cells in upper surfaces of the Transwell chambers were wiped with cotton swabs and the migrated cells remained on the chamber’s bottom surface were fixed with 4% paraformaldehyde and stained with Giemsa solution. The stained migrated cells

The real-time reverse transcriptase PCR was used to detect SET8 mRNA expression in PC cells after treatment with curcumin or microRNA-7 mimics transfection. Total RNA was extracted using Trizol reagent and 2 mg of total RNA was reverse transcribed using First Strand cDNA Synthesis Kit (Fermentas). Then the real-time quantitative PCR was performed following manufacturer’s protocol, using SYBR Green Assay kit (TaKaRa). The relative expression was analyzed using the comparative Ct method by the 7500 Real-Time System software.

Fig. 5. Effect of miR-7 inhibitor on invasion in PC cell. (A and B) Cell invasion was measured using Transwell inserts with Matrigel in AsPC-1 (A) and BxPC-3 (B) cells after curcumin treatment or miR-7 inhibitor or the combination. *P < 0.05 compared to control; **P < 0.05 compared to curcumin treatment alone or miR-7 inhibitor alone.

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2.8. MiR-7 mimics transfection The AsPC-1 and BxPC-3 cells were transfected with miR-7 mimics (GenePharma, Shanghai, China) or the nonspecific control using lipofectamine RNAiMAX reagent (Invitrogen) following the manufacturer’s protocol. MiR-7 mimics: sense 50 -UGG AAG ACU AGU GAU UUU GUU GU-30 ; antisense 50 -AAC AAA AUC ACU AGU CUU CCA UU30 . After the indicated periods of incubation, the cells were subjected to further analysis as presented under the Section 3. 2.9. MiRNA-7 inhibitor transfection Cells were seeded in six-well plates and transfected with antisense miR-7 olignucleotide (GenePharma, Shanghai, China) or

the nonspecific control using DharmaFect Transfection Reagent (Dharmacon, CO) as described previously (Chen et al., 2014). MiR-7 inhibitor: 50 -ACA ACA AAA AUC ACU AGU CUU CCA-30 .

2.10. Western blotting assay Western blotting was performed as described previously (Yang et al., 2014). The cells were lysed in cold lysis buffer with freshly added protease inhibitors. The protein concentrations were determined by BCA protein assay. The proteins were separated by SDS-PAGE and subsequently transferred to a PVDF membrane. The membrane was blocked with TBST containing 5% nonfat milk and immunoblotted with indicated antibodies.

Fig. 6. Effect of curcumin and miR-7 on SET8 expression. (A) The SET8 mRNA level was detected by real-time RT-PCR in AsPC-1 and BxPC-3 cells after curcumin treatment or miR-7 mimics or the combination. (B) The SET8 expression was detected by Western blotting analysis in AsPC-1 and BxPC-3 cells after curcumin treatment or miR-7 mimics or the combination. (C) The SET8 expression was detected by Western blotting analysis in AsPC-1 and BxPC-3 cells after curcumin treatment or miR-7 inhibitor or the combination.

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2.11. Statistical analysis All statistical analyses were conducted using GraphPad Prism 4.0 (Graph Pad Software, La Jolla, CA). Results were expressed as means  SD. Student test was used to evaluate statistical significance. P < 0.05 was considered as statistically significant.

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proliferation, we performed anti-miR-7 transfection studies. In line with this finding, we observed that inhibition of miR-7 promoted cell growth in PC cells (Fig. 3C). Moreover, down-regulation of miR-7 partly abrogated cell growth inhibition induced by curcumin treatment (Fig. 3C). 3.6. MiR-7 mimics triggered apoptosis in PC cells

3. Results 3.1. Curcumin inhibited cell proliferation in PC cells First, we conducted MTT assay to measure cell survival of AsPC1 and BxPC-3 cells after curcumin treatment for 72 h. Our results showed curcumin treatment caused cell proliferation inhibition in a dose-dependent manner in both AsPC-1 and BxPC-3 cells (Fig. 1A). The IC50 value of curcumin after 72 h of incubation was 6 mM and 3 mM for AsPC-1 and BxPC-3 cells, respectively. In the following studies, we chose IC50 for further investigation. 3.2. Curcumin induced cell apoptosis in PC cells We further measured apoptotic cell death using an Annexin V-FITC/PI apoptosis detection kit to analyze whether curcumin could induce apoptosis, leading to cell growth inhibition. As shown in flow cytometer results, the percentage of apoptosis cells was increased by curcumin treatment in a dose-dependent manner in both AsPC-1 and BxPC-3 cells compared with the control group (Fig. 1B and C). Specifically, 3 mM and 6 mM curcumin induced apoptosis from 6.5% to 12.7% and 24.9% in AsPC-1 cells, respectively (Fig. 1B). Consistently, 1.5 mM and 3 mM curcumin triggered apoptosis from 5.5% to 24.1% and 43.3% in BxPC-3 cells, respectively (Fig. 1C). Moreover, our confocal scanning laser microscopy results also revealed that curcumin treatment induced cell apoptosis in both PC cells (Fig. 1D). 3.3. Curcumin inhibited cell migration and invasion in PC cells We performed a scratch wound-healing assay and Transwell assay to detect the effect of curcumin on the cell migration of AsPC-1 and BxPC-3 cells. As expected, we found that curcumin significantly inhibited the migration of BxPC-3 and AsPC-1 cells compared with the control groups (Fig. 2A, 2B). Furthermore, the Transwell assay showed that curcumin decreased the invasion of these two PC cells (Fig. 2C,D). These results clearly suggest that curcumin can inhibit migration and invasion in PC cells. 3.4. Curcumin increased miR-7 expression in PC cells It has been reported that miR-7 plays an important role in tumorigenesis including PC (Kalinowski et al., 2014). To investigate whether curcumin treatment could regulate the expression of miR-7 in PC cells, we performed miRNA RT- PCR assay. As shown in Fig. 3A, miR-7 expression was significantly increased after curcumin treatment in both AsPC-1 and BxPC-3 cells. Next, we explore the effects of miR-7 on cell growth, apoptosis, migration, and invasion in PC cells. 3.5. MiR-7 mimics inhibited cell growth in PC cells To detect the function of miR-7 in cell proliferation, we transfected PC cells with miR-7 mimics for 72 h followed by MTT assay. We found that re-expression of miR-7 inhibited cell proliferation in both AsPC-1 and BxPC-3 cells (Fig. 3B). Consistently, miR-7 mimics and curcumin treatment caused more significant inhibition than miR-7 mimics alone or curcumin treatment alone (Fig. 3B). To further explore the effects of miR-7 on cell

We also measured whether re-expression of miR-7 induced cell apoptosis in PC cells. Indeed, we observed that miR-7 mimics increased percentage of apoptotic cells in AsPC-1 and BxPC-3 cells (Fig. 4A and B). The combination of miR-7 transfection and curcumin treatment showed higher percentage of apoptotic cells than miR-7 mimics alone and curcumin alone (Fig. 4A and B). These results suggested that curcumin induced cell apoptosis partly due to up-regulation of miR-7 in PC cells. 3.7. MiR-7 mimics inhibited invasion in PC cells To evaluate the contribution of miR-7 to cell invasive capacity in both AsPC-1 and BxPC-3 cells, we performed Transwell invasion chamber assay to examine the invasion potential of miR-7 mimics transfected cells. Our findings indicated that cell invasive capacity was decreased by miR-7 mimics treatment in PC cells (Fig. 4C and D). Notably, miR-7 mimics and curcumin treatment caused a lower level of penetration through the membrane compared with miR-7 mimics alone or curcumin treatment alone (Fig 4C and D). Consistent with these results, we observed that inhibition of miR-7 enhanced cell invasion in PC cells (Fig. 5A and B). Furthermore, miR-7 inhibitor rescued curcumin-induced cell invasion inhibition to a certain degree (Fig.5A and B). 3.8. Curcumin and miR-7 mimics inhibited SET8 expression in PC cells It has been reported that miR-7 suppressed epithelial–mesenchymal transition and invasion of breast cancer cells through targeting histone methyltransferase SET8 (Yu et al., 2013). Therefore, we further explored whether curcumin could inhibit the expression of SET8, one of miR-7 targets, in PC cells. We conducted real-time RTPCR to detect SET8 mRNA level in PC cells treated with curcumin and miR-7 mimics. We observed that curcumin decreased the SET8 mRNA in AsPC-1 and BxPC-3 cells (Fig. 6A). Notably, we found that miR-7 mimics also decreased SET8 mRNA in PC cells. Moreover, curcumin plus miR-7 mimics decreased SET8 mRNA to a greater degree compared to curcumin alone (Fig. 6). Additionally, results from the Western blotting analysis demonstrated that both curcumin and miR-7 mimics inhibited the expression of SET8 in PC cells (Fig. 6B). Consistent with the note that p53 is one of SET8 downstream targets (Dhami et al., 2013), we observed that miR7 mimics led to upregulation of p53 expression, resulting in downregulation of Bcl-2 expression that is one of p53 downstream effectors (Hemann and Lowe, 2006) (Fig. 6B). Curcumin and miR7 mimics together inhibited the expression of SET8 to minimum compared with curcumin alone or miR-7 mimics alone (Fig. 6B). In support of role of miR-7 in regulation of SET8, we also measured the SET8 expression in PC cells after inhibition of miR-7. We found that miR-7 inhibitor treatment led to elevated SET8 and Bcl-2 levels and reduced p53 expression (Fig. 6C). These results suggest that curcumin exerts its anticancer effects partly through up-regulation of miR-7 and subsequent down-regulation of SET8 and control of its target genes in PC cells. 4. Discussion A growing body of data implicates that curcumin exerts its inhibitory effects on the cancer development and progression via

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regulating miRNAs. For example, it has been reported that curcumin modulated Akt/FOXO1 axis via upregulation of miR-9 in ovarian cancer cells (Zhao et al., 2014). Moreover, curcumin was found to induce apoptosis in multidrug-resistant human lung adenocarcinoma cells through down-reuglation of miR-186 (Zhang et al., 2010). Similarly, curcumin inhibited metastasis through governing miR-181b expression and its targets CXCL1 (chemokine (C-X-C motif) ligand 1) and CXCL2 in breast cancer (Kronski et al., 2014). Consistently, curcumin regulated the expression of Src-Akt axis via modulating miR-203 level in bladder cancer (Saini et al., 2011). Several studies have revealed that curcumin regulated multiple other miRNAs such as miR-200 (Liang et al., 2013; Saini et al., 2011), miR-34 (Roy et al., 2012), and miR-19 (Li et al., 2014). In the current study, we provide the evidence showing that curcumin inhibited cell growth and invasion through up-regulating miR-7 in PC cells. A number of observations showed that miR-7 could serve as tumor suppressor gene in a variety of human cancers (Kalinowski et al., 2014). To support this concept, reduced levels of miR-7 have been associated with the development of cancer and metastasis. Consistently, miR-7 inhibited tumor growth and metastasis and increased the sensitivity of drug resistant cancer cells to chemotherapeutic agents (Kalinowski et al., 2014). Specifically, miR-7 inhibited tumor metastasis and reversed EMT via Akt/ERK1/ 2 (extracellular regulated protein kinases) inactivation by reducing EGFR (epidermal growth factor receptor) expression in ovarian cancer (Zhou et al., 2014) and gastric cancer (Xie et al., 2014). In support of role of miR-7, it has been found that miR-7 suppressed cell proliferation and induced apoptosis by targeting XRCC2 (X-ray repair complementing defective repair in Chinese hamster cells 2) in colorectal cancer (Xu et al., 2014). Consistent with these evidences, miR-7 caused G1 cell cycle arrest by targeting cyclin E1 in hepatocellular carcinoma cells (Zhang et al., 2014). These findings argued that up-regulation of miR-7 could represent a new treatment approach for human cancer. To further support the role of miR-7, we observed that up-regulation of miR-7 inhibited cell proliferation, migration and induced cell apoptosis in PC cells. More importantly, we found that curcumin significantly increased the expression of miR-7, suggesting that curcumin could be a potential agent to treat PC through modulating miR-7 expression. It has been shown that SET8, one of miR-7 targets, plays an essential role in tumorigenesis (Yu et al., 2013). It is clear that SET8, also known as PR-Set7/9, SETD8, KMT5A, targets H4K20 for monomethylation. Recently, studies have revealed that SET8 promoted EMT and enhanced the invasive potential of breast cancer cells (Yang et al., 2012). Furthermore, SET8 regulates the function of PCNA (proliferating cell nuclear antigen) protein through lysine methylation, leading to promoting carcinogenesis (Takawa et al., 2012). Consistently, overexpression of SET8 was observed in various types of human cancers including PC (Takawa et al., 2012). SET8 was also found to methylate p53, leading to inhibition of p53-mediated transcription activation of target genes (Shi et al., 2007). Moreover, SET8 was found to methylate Numb, resulting in increased p53 ubiquitination and degradation (Dhami et al., 2013). In line with this report, we observed that curcumin inhibited SET8 expression and subsequently increased p53 expression and decreased Bcl-2 level. Since SET8 plays an oncogenic role in tumorigenesis, SET8 could be a potential target for intervention of human cancer. To this end, SET8 inhibitors have been developed to induce cell death in leukemia cells (Valente et al., 2012). Our results demonstrated that curcumin could inhibit the expression of SET8 in PC cells, suggesting that curcumin could be a potential inhibitor of SET8. It is important to note that the therapeutic use of curcumin is hampered due to its poor absorption and rapid metabolism in the liver and intestinal wall. For example, less than 5 mg/ml in blood

was detected in rats after oral administration of 400 mg curcumin (Ravindranath and Chandrasekhara, 1980). In humans after a dose of 2 g curcumin, serum levels are either undetectable or very low (Shoba et al., 1998). Patients with PC who received 8 g curcumin by mouth daily have 22–41 ng/ml curcumin at peak in plasma (Dhillon et al., 2008). Current reports have indicated that 2–20 mM curcumin for in vitro studies, 50–200 mg/kg curcumin for mice studies, 8–12 g/day for human trial were typically utilized (Kanai, 2014; Prasad et al., 2014a,b). Due to lack of bioavailability, it is necessary to explore how to enhance the bioavailability of curcumin to reduce the dose of curcumin. Since this compound has a broad range of biological activities, the need to increase bioavailability is widely appreciated. To overcome its low bioavailability, some analogues such as CDF have been synthesized and exhibited anti-tumor activity more effectively than its parent compound curcumin (Dandawate et al., 2012; Padhye et al., 2009). Additionally, various nanotechnology-based formulations of curcumin such as “nanocurcumin” have been developed to overcome the low bioavailability and stability (Bhawana et al., 2011; Bisht et al., 2007; Shehzad et al., 2014). In conclusion, our present study revealed that miR-7 plays a critical role in governing cell growth and apoptosis in PC cells. Our findings also demonstrated that curcumin suppressed cell proliferation, induced cell apoptosis, inhibited cell migration partly through up-regulation of miR-7 and subsequent downregulation of SET8 and its downstream effects including p53. Although it is a proposing tactics to develop SET8 inhibitors, crucumin with nontoxicity could be a potential therapeutic agent in PC treatment. Conflict of interest The authors declare no conflict of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgements This work was supported by grant from National Natural Science Foundation of China (81172087) and the priority academic program development of Jiangsu Higher Education Institutions. This work was also supported in part by the Natural Science Research key Project of Education Office of Anhui Province (KJ2012A196 and KJ2014A153). References Babae, N., Bourajjaj, M., Liu, Y., Van Beijnum, J.R., Cerisoli, F., Scaria, P.V., Verheul, M., Van Berkel, M.P., Pieters, E.H., Van Haastert, R.J., Yousefi, A., Mastrobattista, E., Storm, G., Berezikov, E., Cuppen, E., Woodle, M., Schaapveld, R.Q., Prevost, G.P., Griffioen, A.W., Van Noort, P.I., Schiffelers, R.M., 2014. Systemic miRNA7 delivery inhibits tumor angiogenesis and growth in murine xenograft glioblastoma. Oncotarget 5, 6687–6700. Bartsch, D.K., Gress, T.M., Langer, P., 2012. Familial pancreatic cancer–current knowledge. Nat. Rev. Gastroenterol. Hepatol. 9, 445–453. Beevers, C.S., Zhou, H., Huang, S., 2013. Hitting the golden TORget: curcumin’s effects on mTOR signaling. Anticancer Agents Med. Chem. 988–994. Bhawana, R.K., Basniwal, H.S., Buttar, H.S., Jain, V.K., Jain, N., 2011. Curcumin nanoparticles: preparation, characterization, and antimicrobial study. J. Agric. Food Chem. 59, 2056–2061. Bisht, S., Feldmann, G., Soni, S., Ravi, R., Karikar, C., Maitra, A., 2007. Polymeric nanoparticle-encapsulated curcumin (nanocurcumin): a novel strategy for human cancer therapy. J. Nanobiotechnol. 5, 3. Chakraborty, C., George, C., Priya Doss, Bandyopadhyay, S., 2013. miRNAs in insulin resistance and diabetes-associated pancreatic cancer: the ‘minute and miracle’ molecule moving as a monitor in the 'genomic galaxy'. Curr. Drug Targets 14, 1110–1117.

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