JNK signalling axis promotes human pancreatic cancer cells proliferation

Biomedicine & Pharmacotherapy 68 (2014) 25–30

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Original article

miR-92a/DUSP10/JNK signalling axis promotes human pancreatic cancer cells proliferation§ Gengsheng He a,b, Lei Zhang c, Qing Li d,*, Longqiu Yang e,* a

Center of Transplant Surgery, The Third Xiangya Hospital, Central South University, Changsha, Hunan 421001, PR China Department of General Surgery, the First Affiliated Hospital, South China University, Hengyang, 421001 Hunan, PR China c Department of Anesthesiology, The Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200011, PR China d Department of Anesthesiology, Jiangsu Provincial Hospital of Integrated Chinese Traditional and Western Medicine, 100, Shizi Street, Hongshan Road, 210028 Nanjing, PR China e Department of Anesthesiology, Zhengzhou Central Hospital Affiliated to Zhengzhou University, 195 Tongbai Road, Zhengzhou City, 450007 Henan Province, PR China b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 October 2013 Accepted 11 November 2013

Pancreatic cancer is one of the most common types of cancers in the whole world with a poor prognosis. Finding out how the cancer form and develop is the most important way to cure this cancer. miRNAs, 21– 22 nucleotides regulatory small non-coding RNAs, have been found to be critical involved in the growth of pancreatic cancer. In this study, we found that miR-92a was up regulated in three kinds of human pancreatic cancer cell lines. There is a correlation between miR-92a and malignant degree of human pancreatic cancer cell lines. Then we found that miR-92a was essential for promoting cell proliferation in human pancreatic cancer. Inhibition of the function of miR-92a repressed the proliferation of pancreatic cancer cells. Further, we found that miR-92a enhanced the activation of JNK signalling pathway by directly targeting the JNK signalling inhibitor DUSP10. DUSP10 is responsible for miR-92a induced JNK signalling and cell proliferation. Altogether, our study showed a miR-92a/DUSP10/JNK signalling pathway that plays an important role in regulating the proliferation of pancreatic cancer cells. ß 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Pancreatic cancer MiR-92a DUSP10 Proliferation

1. Introduction Pancreatic cancer is one of the most common causes of tumorrelated death in the world [1,2]. Pancreatic cancer patients have poor prognosis. Few of them who undergo curative resection can alive after 5 years [3–5]. There have been many studies about the mechanism of the formation and development of pancreatic cancer, but they are still not enough. Finding out the factors that can be the chief regulator of pancreatic cancer formation and development will be the most efficacious way to cure the cancer. miRNAs, small non-coding RNA gene products of approximately 22 nucleotides, have been shown to play an important role in regulating the growth of many cancers [6–8]. They have been reported to be critically involved in invasion, migration and viability of many cancers [9,10]. miRNAs influence the translation and degradation of mRNAs by directly target on the partially complementary sites of the 30 untranslated regions (UTR) of

§ This research was supported by the National Natural Science Foundation of China (81300934) and Science and Technology Planning Project of Hunan Province, China (2012FJ3105). * Corresponding authors. E-mail addresses: [email protected] (Q. Li), [email protected] (L. Yang).

0753-3322/$ – see front matter ß 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biopha.2013.11.004

mRNAs [11–13]. The regulating mechanism of most of the miRNAs are still not fully clear, but more and more studies have shown the important function in various processes of pancreatic cancer growth [14–18]. The c-Jun NH2-terminal kinase (JNK) pathway has been reported to be closely related to the formation and development of cancers [19–22]. Mice lack of JNK1 exhibited a down-regulation of carcinogenesis in gastric cancer and hepatocellular cancer [23– 26]. JNK2-deficient mice showed repressed growth of skin tumors [27]. Previous study showed that activation of JNK promotes development of pancreatic cancer. Inhibiting JNK brings about the cancer growth inhibition, cell cycle arrest, and decreased angiogenesis. Inhibition of the activation of JNK might be the therapeutic strategy of pancreatic cancer to prolong the patient survival [19]. In our study, we investigated the underlying mechanism of pancreatic cancer cell rapid proliferation. We found that miR-92a targeted DUSP10 to promote JNK signalling and promote pancreatic cancer cell proliferation. miR-92a/DUSP10/JNK signalling pathway regulates the proliferation of pancreatic cancer cells, indicating that modulation of the activation of miR-92a/DUSP10/ JNK signalling can be one of the targets of pancreatic cancer therapeutic strategy.

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2. Materials and methods 2.1. Cell culture BxPC3, HPAC, MIAPaCa and HEK293 cells were cultured at 37 8C in DMEM medium, supplemented with 10% fetal bovine serum (FBS), 100 mg/mL of streptomycin sulfate 100 U/mL of penicillin sodium, maintained in 5% CO2. 2.2. qRT-PCR for miRNA qRT-PCR was performed by using SYBR Premix Ex TaqTM (Takara) in Mx3000P system (Stratagene). RNA was isolated using the RNAios plus (Takara). miRNA was reverse transcribed to cDNA by using the stem-loop reverse transcription primer (RuiboBio, China). The U6 small nuclear RNA was used as internal controls for qRT-PCR. miRNA qRT-PCR primers were also from RuiboBio Co., Ltd. The amount of gene expression (2-DDCt) was normalized using the U6 reference. 2.3. qRT-PCR for mRNA By using RNAiso plus (Takara), the total RNA was isolated. mRNA was subsequently reverse-transcribed to cDNA by M-MLV Reverse Transcriptase (Promega). PCR included 40-cycles of amplification in Mx3000P system (Stratagene). Expression of target genes (2-DDCt) was normalized against endogenous GAPDH. qRT-PCR primer: DUSP10 PF: 50 -ATCGGCTACGTCATCAACGTC-30 , 50 -TCATCCGAGTGTGCTTCATCA-30 GAPDH PF: 50 -ACAACTTTGGTATCGTGGAAGG-30 , 50 -GCCATCACGCCACAGTTTC-30 2.4. Over-expression vector of DUSP10 DUSP10-over-expressing vector were constructed by inserting the whole-length CDS cloned into Fugw vector. 2.5. MTT assay Cells were cultured in 96-well plates about 1.0  104 cells/mL, exposing to fresh media every other day. During the last 3 h of each day of culture, the cells were treated with methyl thiazolyl tetrazolium (MTT, 50 mg per well, Sigma, USA), dissolved the generated formazan in DMSO for half an hour then record the absorbance at 490 nm. 2.6. Bromodeoxyuridine assay

then incubated with primary DUSP10 antibody (ab140123) for 4 8C overnight. Next day, we incubated the protein with secondary antibodies. GAPDH antibody (ab9485) detected the endogenous GAPDH that was used as loading control. 2.8. Statistical analysis Error bars represent the standard deviation (SD) of three independent experiments. Data was evaluated by Student’s t test. Statistical significance was set as *P < 0.05, **P < 0.01, ***P < 0.001. 3. Results 3.1. miR-92a is up regulated in human pancreatic cancer cells We chose three human pancreatic cancer cell lines including BxPC3, HPAC, MIAPaCa to analyze the levels of miRNA-92a compared with the normal human cell lines HEK 293.We found that the level of miRNA-92a is higher in the pancreatic cancer cell lines, especially in the BxpC3 cells with the highest malignant degree among three cancer cell lines (Fig. 1). This result indicated that there is a positive correlation between miR-92a and malignance of human pancreatic cancer cells. 3.2. miR-92a plays an important role in promoting cell proliferation in human pancreatic cancer To explore the function of miR-92a in human pancreatic cancer cells, we transfected miR-92a inhibitor, a single chain of nuclear acid with reverse complementary sequence of the miR-92a, into the BxpC3 cells. We detected the effect of miR-92a on downregulating the level of miR-92a (Fig. 2A). MTT assay showed that proliferation of BxpC3 cells was reduced by miR-92a inhibition (Fig. 2B). Brdu assay further confirmed that down-regulation of miR-92a significantly impaired the proliferation of BxpC3 cells (Fig. 2C). Then we transfected the miR-92a mimics into the BxpC3 cells (Fig. 2D). Contrary to the effects of miR-92 inhibition, miR-92a over-expression promotes the proliferation of BxpC3 cells by MTT assay (Fig. 2E) and Brdu assay (Fig. 2F). These results indicated that highly expression of miR-92a contributes to the enhanced proliferation of human pancreatic cancer cells. 3.3. miR-92a enhances the activation of JNK signalling pathway Previous study showed that JNK is frequently activated in human pancreatic cancer and associated with the cell cycle

Bromodeoxyuridine (Brdu) assay was performed by using a competitive colourimetric cell proliferation ELISA assay kit (Roche Diagnostics, Mannheim, Germany). Seeded the cells at 4  103 cells per well in 100 mL culture medium. Cultured the cells for 24 hours. Then cells were added with 10 mL bromodeoxyuridine (BrdU) labeling solution for 7 h. According to the manufacturer’s instructions, the absorbance was measured within five minutes at 450 nm/620 nm. 2.7. Western blotting Cells were lysed in 1  lysis buffer that was diluted from 5  lysis buffer. 5  lysis buffer contains (0.5 mol/L Tris-HCl (pH 6.8) 2.5 mL, DTT 0.39 g, SDS 0.5 g, bromophenol blue 0.025 g, glycerine 2.5 mL) and kept on ice for 10 min. Equal amount of protein was loaded. Nitrocellulose membrane was used for accepting the protein transferring onto it. The membrane was

Fig. 1. miR-92a is up-regulated in human pancreatic cancer cells. miRNA-92a is highly expressed in the pancreatic cancer cell lines, especially in the BxpC3 cells with the highest malignant degree among three cancer cell lines. For all experiments n = 3, average  SD; Student’s t-test. *P < 0.05, **P < 0.01,***P < 0.001.

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Fig. 2. miR-92a promotes cell proliferation in human pancreatic cancer. (A) miR-92a inhibitor down-regulated the level of miR-92a in the BxpC3 cells. (B) miR-92a inhibitor reduced the proliferation of BxpC3 cells in MTT assay. (C) miR-92a inhibitor significantly impaired the proliferation of BxpC3 cells in Brdu assay.(D) miR-92a mimics were transfected into the BxpC3 cells. (E) miR-92a mimics promoted the proliferation of BxpC3 cells in MTT assay. (F) miR-92a mimics significantly impaired the proliferation of BxpC3 cells in Brdu assay. For all experiments n = 3, average  SD; Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

regulation. We next tested whether JNK signalling pathway is involved in miR-92a induced proliferation of BxpC3 cells. The level of phosphorylated JNK in BxpC3 cells transfected by the miR92a mimics or control mimics was detected by Western blot. miR92a significantly up-regulates the level of phosphorylated JNK(Fig. 3A). Further, we inhibited the function of miR-92a by transfecting the miR-92a inhibitor into the BxpC3 cells. In contrary to the transfection of miR-92a, cells transfected with miR-92a inhibitor showed lower level of phosphorylated JNK (Fig. 3B). These results confirmed that miR-92a activates JNK signalling in BxpC3 cells. 3.4. miR-92a directly targets DUSP10, a JKN signalling inhibitor, in BxPC3 cells To understand how miR-92a is linked to JKN signalling, we screened the target genes of miR-92a by bioinformatic tools.

Using target prediction program Targetscan, Miranda and miRBase, we found that DUSP10, a gene associated with the regulation of JKN signalling, can be directly targeted by miR-92a (Fig. 4A). Further, we found that DUSP10 expression in BxPC3, HPAC, MIAPaCa and HEK293 showed a negative correlation with the miR-92a (Fig. 4B). Wild-type and mutant 30 UTR reporter vector of DUSP10 gene were constructed. The mutant construct contains a seven base pairs deletion of miR-92a binding sites. As shown in Fig. 4C, miR-92a repressed the luciferase activity of the wild-type DUSP10 30 UTR compared to the group transfected with miRNA control, while miR-92a had no effect on mutant DUSP10 reporter activity. In order to know whether miR-92a down-regulates the endogenous DUSP10, we transfected the miR-92a into BxPC3 cells and found that both mRNA and protein levels of DUSP10 were significantly down-regulated (Fig. 4D, E).These results suggest that miR-92a regulates the activation of JNK signalling through DUSP10.

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Fig. 3. miR-92a enhances the activation of JNK signaling pathway. (A) miR-92a significantly up-regulates the level of phosphorylated JNK. (B) miR-92a inhibitor downregulated the level of phosphorylated JNK. For all experiments n = 3, average  SD; Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4. miR-92a directly targets DUSP10, a JKN signaling inhibitor, in BxPC3 cells. (A) DUSP10 can be directly targeted by miR-92a. (B) The expression of DUSP10 in BxPC3, HPAC, MIAPaCa and HEK293. (C) miR-92a repressed the luciferase activity of the wild-type DUSP10 3’ UTR while miR-92a had no effect on mutant DUSP10 reporter activity. (D) miR-92a significantly down-regulated DUSP10 in mRNA level. (E) miR-92a significantly down-regulated DUSP10 in protein level. For all experiments n = 3, average  SD; Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

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Fig. 5. miR-92a targets DUSP10, which is responsible for the JNK phosphorylation and cell proliferation. (A) over-expression of DUSP10 rescued miR-92a-induced reduction of DUSP10 in mRNA level in BxPC3 cells. (B) over-expression of DUSP10 rescued miR-92a-induced reduction of DUSP10 in protein level. (C) Over-expression of DUSP10 rescued miR-92a-induced decrease of phosphorylation of JNK. (D) Over-expression of DUSP10 rescued miR-92a-induced the promotion of proliferation in MTT assay. (E) Overexpression of DUSP10 rescued miR-92a-induced the promotion of proliferation in Brdu assay. For all experiments n = 3, average  SD; Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

3.5. miR-92a targets DUSP10, which is responsible for the JNK phosphorylation and cell proliferation To test whether DUSP10 mediates the miR-92a induced activation of JNK signalling and cell proliferation in BxPC3 cells, we performed the gain and off assay to investigate whether DUSP10 can rescue the promotion of proliferation mediated by miR-92a. BxPC3 cells were transfected with miR-92a mimics or miRNA control. Six hours later, these cells were over-expressed with DUSP10 or empty vector. Western blot and qRT-PCR analysis showed that over-expression of DUSP10 rescued miR-92a-induced reduction of DUSP10 48 h after transfection (Fig. 5A, B).

Phosphorylation of JNK decreased by DUSP10 (Fig. 5C). MTT (Fig. 5D) and Brdu (Fig. 5E) assay confirmed that DUSP10 rescued the promotion of miR-92a in proliferation of BxPC3 cells. These results showed that miR-92a can target DUSP10 to induce JNK phosphorylation and promote cell proliferation. 4. Discussion In this study, we found that miR-92a/DUSP10/JNK signalling pathway plays an important role in regulating the proliferation of pancreatic cancer cells. These results indicate that intervening the activation of miR-92a/DUSP10/JNK axis regulates the growth of

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pancreatic cancer cells, which might be one of the pancreatic cancer therapeutic strategies. Rapid proliferation is the one of most important characteristics of the cancer cells. Studies have been already finding the mechanisms of rapid proliferation of cancer cells [28,29]. But rapid proliferation is such a multistep process that the mechanisms still remain largely unclear. miRNA has been reported to be the regulator of the cancer growth. In our study we found that miR-92a is up regulated in human pancreatic cancer cells. We also found that the most malignant pancreatic cancer cell line BxpC3 had the highest expression level of miR-92a, which indicates that miR-92a may be the marker of malignant degree of pancreatic cancer cells. Further, we determined that the miR-92a promotes the activation of JNK signalling by directly targeting the DUSP10, an inhibitor of JNK signalling. JNK signalling is the well known signalling that regulates the formation and development of cancers [19–22]. JNK is related to the oncogenic transformation. But the regulating mechanism of JNK and the upstream regulator of JNK remain unclear [20]. In our study, we confirmed the function of JNK signalling in pancreatic cancer cells. We found that miR-92a unregulated the activation of JNK signalling. Inhibiting miR-92a could down-regulate the activation of JNK signalling. These results showed that miR-92a is the upstream regulator of JNK signalling. Further, we found that miR-92a targeted DUSP10 to regulate the JNK signalling. The expression of DUSP10 is the lowest in BxpC3 cells, which is related to the highest level of miR-92a. Finally we determined that miR92a/DUSP10/JNK signalling forms an axis to regulate the proliferation of pancreatic cancer. Our study showed a miR-92a/DUSP10/JNK signalling axis, which may be used as one of the pancreatic cancer therapeutic strategies. Additionally, the expression level of miR-92a or DUSP10 may be a marker to predict the outcome of pancreatic cancer during the early stage.

[6] [7]

[8] [9] [10]

[11] [12] [13] [14]

[15]

[16]

[17] [18]

[19]

[20] [21] [22]

[23]

Disclosure of interest [24]

The authors declare that they have no conflicts of interest concerning this article.

[25]

References [26] [1] Hirata K, Egawa S, Kimura Y, Nobuoka T, Oshima H, Katsuramaki T, et al. Current status of surgery for pancreatic cancer. Dig Surg 2007;24:137–47. [2] Jemal A, Murray T, Samuels A, Ghafoor A, Ward E, Thun MJ. Cancer statistics, 2003. CA Cancer J Clin 2003;53:5–26. [3] Hawes RH, Xiong Q, Waxman I, Chang KJ, Evans DB, Abbruzzese JL. A multispecialty approach to the diagnosis and management of pancreatic cancer. Am J Gastroenterol 2000;95:17–31. [4] Matsuno S, Egawa S, Fukuyama S, Motoi F, Sunamura M, Isaji S, et al. Pancreatic cancer registry in Japan: 20 years of experience. Pancreas 2004;28:219–30. [5] Kenoki Ohuchida, Kazuhiro Mizumoto, Cui Lin, Hiroshi Yamaguchi, Takao Ohtsuka. et al. MicroRNA-10a is overexpressed in human pancreatic cancer

[27] [28]

[29]

and involved in its invasiveness partially via suppression of the HOXA1 gene. Ann Surg Oncol 2012;19:2394–402. Garzon R, Fabbri M, Cimmino A, Calin GA, Croce C. MicroRNAs expression and function in cancer. Trends Mol Med 2006;12:580–7. Bracken CP, Gregory PA, Khew-Goodall Y, Goodall GJ. The role of microRNAs in metastasis and epithelial-mesenchymal transition. Cell Mol Life Sci 2009;10:1682–99. Esquela-Kerscher A, Slack FJ. Oncomirs – microRNAs with a role in cancer. Nat Rev Cancer 2006;6:259–69. Liu T, Tang H, Lang Y, Liu M, Li X. MicroRNA-27a functions as an oncogene in gastric adenocarcinoma by targeting prohibitin. Cancer Lett 2009;273:233–42. Huang Q, Gumireddy K, Schrier M, le Sage C, Nagel R, et al. The microRNAs miR373 and miR-520c promote tumour invasion and metastasis. Nat Cell Biol 2008;10:202–10. Lee RC, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans. Science 2001;294:862–5. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. Identification of novel genes coding for small expressed RNAs. Science 2001;294:853–8. Lau NC, Lim LP, Weinstein EG, Bartel DP. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 2001;294:858–62. Wang WS, Liu LX, Li GP, et al. Combined serum CA19-9 and miR-27a-3p in peripheral blood mononuclear cells to diagnose pancreatic cancer. Cancer Prev Res (Phila) 2013;6:331–8. Baraniskin A, No¨pel-Du¨nnebacke S, Ahrens M, et al. Circulating U2 small nuclear RNA fragments as a novel diagnostic biomarker for pancreatic and colorectal adenocarcinoma. Int J Cancer 2013;132:E48–57. Morimura R, Komatsu S, Ichikawa D, et al. Novel diagnostic value of circulating miR-18a in plasma of patients with pancreatic cancer. Br J Cancer 2011;105:1733–40. Liu J, Gao J, Du Y, et al. Combination of plasma microRNAs with serum CA19-9 for early detection of pancreatic cancer. Int J Cancer 2012;131:683–91. Liu R, Chen X, Du Y, et al. Serum microRNA expression profile as a biomarker in the diagnosis and prognosis of pancreatic cancer. Clin Chem 2012;58: 610–8. Takahashi R, Hirata Y, Sakitani K, Nakata W, Kinoshita H, et al. Therapeutic effect of c-Jun N-terminal kinase inhibition on pancreatic cancer. Cancer Sci 2013;104:337–44. Kennedy NJ, Davis RJ. Role of JNK in tumor development. Cell Cycle 2003;2:199–201. Nateri AS, Spencer-Dene B, Behrens A. Interaction of phosphorylated c-Jun with TCF4 regulates intestinal cancer development. Nature 2005;437:281–5. Yunoki T, Kariya A, Kondo T, Hayashi A, Tabuchi Y. The combination of silencing BAG3 and inhibition of the JNK pathway enhances hyperthermia sensitivity in human oral squamous cell carcinoma cells. Cancer Lett 2013;335:52–7. Shibata W, Maeda S, Hikiba Y, et al. c-Jun NH2-terminal kinase 1 is a critical regulator for the development of gastric cancer in mice. Cancer Res 2008;68: 5031–9. Sakurai T, Maeda S, Chang L, Karin M. Loss of hepatic NF-kappa B activity enhances chemical hepatocarcinogenesis through sustained c-Jun N-terminal kinase 1 activation. Proc Natl Acad Sci USA 2006;103:10544–51. Hui L, Zatloukal K, Scheuch H, Stepniak E, Wagner EF. Proliferation of human HCC cells and chemically induced mouse liver cancers requires JNK1-dependent p21 downregulation. J Clin Invest 2008;118:3943–53. Chen F, Castranova V. Beyond apoptosis of JNK1 in liver cancer. Cell Cycle 2009;8:1145–7. Chen N, Nomura M, She QB, et al. Suppression of skin tumorigenesis in c-Jun NH(2)-terminal kinase-2-deficient mice. Cancer Res 2001;61:3908–12. Harald Ehrhardt, Simone Fulda, Irene Schmid, John Hiscott, laus-Michael Debatin, Irmela Jeremias. TRAIL induced survival and proliferation in cancer cells resistant towards TRAIL-induced apoptosis mediated by NF-B. Oncogene 2003;22:3842–52. Mapara MY, Bargou R, Zugck C, Dohner H, Ustaoglu F, Jonker RR, et al. APO-1 mediated apoptosis or proliferation in human chronic B lymphocytic leukemia: correlation with bcl-2 oncogene expression. Eur J Immunol 1993;23: 702–8.