MicroRNA Signaling Pathway Network in Pancreatic Ductal Adenocarcinoma

Accepted Manuscript MicroRNA Signaling Pathway Network in Pancreatic Ductal Adenocarcinoma Longhao Sun, Corrine Ying Xuan Chua, Weijun Tian, Zhixiang Zhang, Paul J. Chiao, Wei Zhang PII:

S1673-8527(15)00133-2

DOI:

10.1016/j.jgg.2015.07.003

Reference:

JGG 385

To appear in:

Journal of Genetics and Genomics

Received Date: 23 May 2015 Revised Date:

16 July 2015

Accepted Date: 22 July 2015

Please cite this article as: Sun, L., Xuan Chua, C.Y., Tian, W., Zhang, Z., Chiao, P.J., Zhang, W., MicroRNA Signaling Pathway Network in Pancreatic Ductal Adenocarcinoma, Journal of Genetics and Genomics (2015), doi: 10.1016/j.jgg.2015.07.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT MicroRNA Signaling Pathway Network in Pancreatic Ductal Adenocarcinoma

Longhao Sun1,4, Corrine Ying Xuan Chua1,3, Weijun Tian4, Zhixiang Zhang4, Paul J.

of Pathology, The University of Texas MD Anderson Cancer Center,

Houston 77030, USA 2Department

of Molecular and Cellular Oncology, The University of Texas MD

Anderson Cancer Center, Houston 77030, USA

University of Texas Graduate School of Biomedical Sciences, Houston 77030,

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3The

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1Department

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Chiao2,3, Wei Zhang1,2,3, *

USA 4Department

of General Surgery, Tianjin Medical University General Hospital,

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Tianjin 300052, China

*Corresponding authors:

Tel: +1 713 745 1103, fax: +1 713 792 5549.

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E-mail: [email protected] (W. Zhang)

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ACCEPTED MANUSCRIPT Abstract Pancreatic ductal adenocarcinoma (PDAC) is considered to be the most lethal and aggressive malignancy with high mortality and poor prognosis. Their responses to current multimodal therapeutic regimens are limited. It is urgently needed to identify the molecular mechanism underlying pancreatic oncogenesis. Twelve core

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signaling cascades have been established critical in PDAC tumorigenesis by governing a wide variety of cellular processes. MicroRNAs (miRNAs) are aberrantly expressed in different types of tumors and play pivotal roles as post-transcriptional regulators of gene expression. Here, we will describe how miRNAs regulate

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different signaling pathways that contribute to pancreatic oncogenesis and progression.

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Keywords: MicroRNA; Signaling pathway; Tumorigenesis; Pancreatic ductal

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adenocarcinoma

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ACCEPTED MANUSCRIPT INTRODUCTION

Pancreatic ductal adenocarcinoma (PDAC) is one of the most common and devastating human malignancies, with a median survival of six months and an overall five-year survival rate of 5%, which makes it the fourth leading cause of cancer-related deaths

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worldwide (Siegel et al., 2014). The major oncogenic hallmarks of PDAC are early and aggressive local invasion, metastasis and resistance to chemotherapy and radiotherapy. Because of the lack of early specific symptoms and signs, patients with PDAC are usually diagnosed at an advanced stage, which precludes curative surgery and leads to a poor

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prognosis. Early detection and diagnosis of PDAC are therefore extremely important to improve patient survival. Routine imaging methods such as ultrasound, computed tomography (CT) or magnetic resonance imaging (MRI) are not sensitive enough in

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early detection of pancreatic cancer (Rosty et al., 2002). Although CA 19-9 is treated as the most valuable biomarker in early diagnosis and prognosis assessment of pancreatic cancer, there is still limitation in clinical applications because of poor sensitivity. Therefore, we need to elucidate the underlying molecular mechanisms of PDAC in order to develop more clinically beneficial and efficient diagnostic and therapeutic strategies,

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especially to detect disease at a potentially curable stage.

MicroRNAs (miRNAs) are endogenous small (19‒24 nucleotides long) non-coding RNAs that play important roles in the negative regulation of gene expression by base pairing

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to complementary sites in the 3’-untranslated region (UTR) of target mRNA causing translational repression or the degradation of the target mRNAs (Croce, 2009; Huang et

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al., 2013). More than 50% of the known miRNAs are aberrantly expressed in different types of tumors, play pivotal roles as post-transcriptional regulators of gene expression and elicit oncogenic or tumor-suppressive functions by directly targeting oncogenes or tumor suppressive genes, respectively (Slack et al., 2008; Sun et al., 2015). Their potential roles in human cancer diagnosis, progression and metastasis are extensively researched and may provide new insights in cancer research (Broderick et al., 2011; Liu et al., 2014).

PDAC progresses due to the accumulation of gene mutations, and many signaling pathways are implicated in PDAC oncogenesis and progression. A recent study 3

ACCEPTED MANUSCRIPT established that 12 core signaling cascades are genetically altered in 67%‒100% PDAC, including KRAS signaling, Hedgehog signaling, apoptosis, control of G1/S phase transition, TGF-β signaling and Wnt signaling (Jones et al., 2008). Many miRNAs can regulate a wide variety of oncogenic biological processes by affecting the essential components of these signaling cascades (Ding, 2014). Here, we will describe how

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miRNAs regulate different signaling pathways that contribute to pancreatic oncogenesis and progression (Table 1).

KRAS SIGNALING

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KRAS is a ~21 kDa intracellular membrane bound protein from the GTPase superfamily which cycles between GTP-bound active and GDP-bound inactive states. KRAS plays a significant role in cell survival, proliferation, and migration through regulation of

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several signal transduction pathways like RAF, MAP2K, MAPK, Tiam1, RalA-GEF and PI3K-AKT. KRAS is a major regulator of pancreatic carcinogenesis, and is crucial in the development and progression of PDAC. Mutation of the KRAS oncogene (mostly KrasG12D) is the most frequent genetic alteration associated with pancreatic cancer as it has been identified in up to 90% of all PDAC cases (di Magliano et al., 2013). Constitutive

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activation of KRAS triggers the persistent activation of downstream signaling cascades, such as the RAF/ERK pathway, the PI3K/AKT pathway, and NF-κB (Pylayeva-Gupta et al., 2011), which consequently affects the induction of sustained proliferation, metabolic reprogramming, angiogenesis, anti-apoptosis, chemoresistance, remodeling of the

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tumor microenvironment, evasion of the immune response, cell migration and metastasis, and its downstream signal pathway that leads to their degradation or

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translational inhibition. Even though KRAS is an increasingly promising therapeutic target in PDAC and other KRAS-driven cancers, efficient therapeutic strategies are still urgently needed. Several miRNAs that directly target KRAS and its downstream signal pathways have been identified recently. In this section we review the essential roles of miR-217, -206, -143/145, -126, -96, and Let-7 in mediating the regulation of oncogenic KRAS in PDAC.

MiR-217 A study by Zhao et al. (2010) showed that miR-217 is frequently down-regulated in the nuclei of the ductal epithelia cells in PDAC tissue specimens and cell lines, demonstrating 4

ACCEPTED MANUSCRIPT the potential tumor-suppressive role of miR-217 in PDAC development. KRAS was predicted to be a potential direct target of miR-217 by in silico prediction, which was further confirmed by dual-luciferase reporter gene analysis. MiR-217 can inhibit the malignant behavior of PDAC by regulating KRAS and KRAS signaling. Overexpression of miR-217 in PDAC cells down-regulates KRAS protein level and reduces the constitutive

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phosphorylation of downstream AKT, resulting in reduced tumor cell growth, anchorage-independent colony formation and in vivo xenograft tumor growth.

MiR-206

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Keklikoglou et al. (2014) revealed that miR-206 is abrogated in human PDAC tissues and cell lines, and acts as a tumor suppressor in PDAC by blocking cell cycle progression, cell proliferation, migration, invasion, tumor lymphangiogenesis, and angiogenesis in vitro

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and in vivo. Furthermore, they have shown that miR-206 directly targets KRAS and annexin A2 (ANXA2), causing a suppressive function on oncogenic KRAS-induced NF-κB transcriptional activity. As a result, the expression and secretion of many proangiogenic, pro-lymphangiogenic, and pro-inflammatory factors such as cytokine interleukin-8 (IL-8), granulocyte macrophage colony-stimulating factor (GM-CSF),

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vascular endothelial growth factor C (VEGFC), chemokines (C-X-C motif) ligand

mechanism.

MiR-143

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1(CXCL1) , and (C-C motif) ligand 2(CCL2) were reduced through an NF-κB-independent

Loss of miR-143 expression has been reported in several cancer types, including PDAC,

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and may be responsible for the highly aggressive and metastatic characteristics of PDAC. The restoration of miR-143 expression reduces the mRNA expression and protein levels of ARHGEF1 (GEF1), ARHGEF2 (GEF2), and KRAS genes through direct targeting of the transcripts. Furthermore, miR-143 also inhibits migration and invasion capacity of Panc-1 cells, as well as metastasis and growth of pancreatic cancer in animal models by decreasing Rho GTPase activity, and MMP2 and MMP9 protein levels, while elevating Ecadherin protein level (Hu et al., 2012).

MiR-126 MiR-126 targets several crucial genes including KRAS, CRK and ADAM9, which are 5

ACCEPTED MANUSCRIPT involved in PDAC progression and metastasis (Frampton et al., 2012). Jiao et al. (2012) revealed that miR-126 is down-regulated in PDAC and inhibits KRAS protein translation by interacting with a ‘‘seedless’’ site within its 3’-UTR. Restoration of miR-126 reduces migration and invasion capacity of PDAC cell lines by directly regulating KRAS. MiR-126 exhibits tumor suppressive effects by targeting ADAM9, which enhances cancer cell

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invasion by modulating tumor-stromal cell interactions (Hamada et al., 2012).

MiR-96

Yu et al. (2010) reported that miR-96 directly targets the KRAS oncogene and acts as a

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tumor-suppressor in PDAC. The expression of miR-96 was consistently down-regulated in the pancreatic cancer tissues compared to normal tissues in human clinical specimens and cancer cell lines. In vitro and in vivo assays established that miR-96 could inhibit

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pancreatic cancer cell proliferation, apoptosis, migration, and invasion by directly inhibiting the KRAS/AKT signal pathway.

Let-7

It is also documented that let-7 family miRNAs are down-regulated in PDAC and

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function as a tumors-suppressor by negatively regulating KRAS and HMGA2 oncogenes, whose up-regulated activity is essential in PDAC progression (Torrisani et al., 2009;

MiR-143/145

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Watanabe et al., 2009).

KRAS not only functions as a target of miRNAs, but also regulates the transcription of

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miRNAs by suppressing its promoter and initiating a tumor-promoting feed-forward loop. MiR-143 and miR-145 establish their anti-proliferative and pro-apoptotic effects by directly targeting and down-regulating KRAS via Ras-responsive element-binding protein (RREB1). They also affect two major KRAS effectors, the MAPK and the PI3K pathways, in PDAC cells by reducing the phosphorylation of ERK1/2 and AKT. As a feedforward regulatory circuit, KRAS leads to transcriptional repression of the miR-143/145 cluster in a RREB1-dependent manner by directly targeting their promoters (Kent et al., 2010).

These findings identify that these miRNAs function as potent regulators of KRAS and 6

ACCEPTED MANUSCRIPT robustly suppress downstream KRAS signaling, such as AKT, MAPK and PI3K. Since activation of KRAS mutations are nearly ubiquitous in PDAC, suppression of KRAS pathway likely plays a central role in suppressing pancreatic tumorigenesis. Restoration of these miRNAs may represent a promising therapeutic strategy for treatment of PDAC

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and other KRAS-driven cancers (Fig. 1).

TGF-β SIGNALING

Transforming growth factor-β (TGF-β) is a ubiquitously pleiotropic cytokine that plays a critical role in a variety of cellular functions such as development, proliferation,

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differentiation, apoptosis, angiogenesis, and homeostasis in nearly all cell types and tissues by activating and regulating a diverse array of genes (Padua et al., 2009). TGF-β pathway also acts as an essential signaling regulator during the progression of cancer in

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a SMAD or non-SMAD dependent manner (Hata et al., 2009; Mu et al., 2012). PDAC is typically characterized by its stromal-predominant, desmoplastic, and hypovascular microenvironment (Hidalgo, 2010). Although the role of TGF-β pathway in tumor progression is currently paradoxical because of its dual function in tumor suppressing or tumor promoting in a cell context-dependent manner, TGF-β can regulate

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proliferation, epithelial-to-mesenchymal transition (EMT), invasion, metastasis, immunosuppression, and stroma-tumor interaction of PDAC (Tian et al., 2011).

TGF-β plays a central role in the network that triggers the process of EMT through

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activating SMAD3/4 (Ozdamar et al., 2005). Several pro-epithelial miRNAs that oppose TGF-β-induced EMT have also been induced through SMAD1/4 (Samavarchi-Tehrani et

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al., 2010). The miR-200 family (miR-429, -200a, -200b, -200c, and-141) and miR-205 have been revealed as EMT-suppressive miRNAs via directly targeting ZEB1 and ZEB2, that are essential downstream effectors of TGF-β mediated EMT (Gregory et al., 2008). Nevertheless, the TGF-β pathway mediated EMT inducers, such as ZEB1, Snail and Twist, also promote a feed forward loop to keep EMT-stabilizing by suppressing specific miRNAs. Two members of miR-200 family, miR-200c and miR-141, are strong EMTsuppressive miRNAs via inducing epithelial differentiation of undifferentiated cancer cells by up-regulation of E-cadherin. Their direct target ZEB1, on the other hand, functions as a crucial promoter of tumor progression by directly suppressing transcription of its own inhibitors miR-141 and miR-200c (Burk et al., 2008). 7

ACCEPTED MANUSCRIPT The miR-200/ZEB1/E-cadherin axis is an essential downstream pathway of TGF-β in promoting the progression of EMT. Harazono et al. (2013) has identified miR-655 as an EMT-suppressive miRNA using a cell-based reporter system that examines CDH1/Ecadherin-promoter activity. Overexpression of miR-655 induces significant morphologic

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changes and prevents cell motility, migration, and invasion in pancreatic cancer cell lines by increasing E-cadherin expression. It directly targets ZEB1 and TGFβR2 which are characterized as the cardinal components of the TGF-β signaling pathway. These molecules exert their effects through inducing EMT, and function as predictors of poor

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survival in several types of cancer (Spaderna et al., 2006, 2008). MiR-655 is also significantly correlated with better prognosis, and thus may be a prognostic marker or

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even a therapeutic target in human cancer.

The phosphorylation and elevated expression level of non-receptor focal adhesion kinase (FAK) play a central role in promoting TGF-β-induced EMT, being responsible for drug resistance to erlotinib. This EMT-associated kinase switch is enhanced by TGF-β, and is associated with decreased levels of the miR-200 family members as well as

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increased expression of mitogen-inducible gene 6 (MIG6). On the contrary, blockage of TGF-β signaling can inhibit or reverse EMT by up-regulating miR-200c and miR-205, down-regulating MIG6 levels, and increasing erlotinib sensitivity. Izumchenko et al. (2014) also concluded that the elevated ratio of MIG6/miR-200 expression is associated

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with erlotinib resistance in cancer cell lines and can be used as a predictor of response

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to erlotinib in vivo.

SMAD4 is a tumor suppressor gene for PDAC. SMAD4 inactivation or loss of expression occurs in 55% of PDAC patients by intra-genic mutation or deletion in alleles. Loss of SMAD4 is a predictor of disease advancement or metastasis (Singh et al., 2011). Mechanistically, SMAD4 inactivation is attributed to the co-expression of SMAD6 and/or SMAD7 (Singh et al., 2011). Hao et al. (2011) demonstrated that miR-483-3p was overexpressed in human PDAC specimens, leading to elevated cell proliferation and colony formation of pancreatic cancer cells in vitro by directly targeting the 3’-UTR of SMAD4 and reducing its protein level. Furthermore, overexpression of miR-483-3p suppresses SMAD4 target genes such as PAI, p21, and Msx2, and inhibits TGF-β8

ACCEPTED MANUSCRIPT mediated EMT process. MiRNA-421 is also aberrantly overexpressed in PDAC cells and functions to promote cell proliferation and colony formation in vitro. MiRNA-421 also directly suppresses SMAD4 and its downstream genes, such as p21, p15 and Id3 (Hao et al., 2011).

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SMAD7 is also reported as a negative regulator of invasion and metastasis of pancreatic cancer cells by antagonising TGF-β signaling pathway through several mechanisms (Bai et al., 2002; Shi et al., 2004; Wang et al., 2009). Down-regulation of SMAD7 is highly correlated with tumor metastasis and poor prognosis in human pancreatic cancer. Zhu

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et al. (2015) established that miR-367 acts as a suppressor of SMAD7 in human pancreatic cancer by direct targeting and suppressing SMAD7 expression at both mRNA and protein levels. MiR-367 expression level is negatively correlated with SMAD7, and

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low level of SMAD7 is associated poor prognosis of PDAC. MiR-367 overexpression could promote the migration and invasion of PANC-1 and BxPC3 cell lines by mediating EMT, but has no significant effects on proliferation. At the molecular level, the promotion was conducted by increasing TGF-β-induced transcriptional activity (Fig. 2).

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HEDGEHOG SIGNALING

The Hedgehog pathway was first discovered in Drosophila and plays a critical role in governing embryological development (van den Brink, 2007). There are three Hedgehog ligands, Indian, Desert and Sonic Hedgehog (Ihh, Dhh, and Shh) that participate in the

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signaling activation in mammalian development. Hedgehog ligands bind to the common receptor Patched1 (PTCH1) to induce receptor internalization and SMO activation

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(Rubin et al., 2006). The effectors of the Hedgehog signaling pathway include Gliomaassociated antigen (Gli) family (Gli1, Gli2 and Gli3) transcription factors (Hui et al., 2011). Activated SMO induces a signaling cascade of Gli transcription factors and their downstream target genes (Pan et al., 2006). MAPK and PI3K signaling cascades are also involved in Hedgehog-mediated cellular responses, leading to enhanced cancer cell proliferation and survival through Gli1 (Dosch et al., 2010). Aberrant expression and secretion of Hedgehog from pancreatic tumors are tightly regulated during tumor initiation and progression of PDAC in a paracrine manner. Elevated expression of Shh has been reported in up to 70% of human PDAC specimens (Thayer et al., 2003). Several miRNAs have been shown to target hedgehog signaling either by targeting the pathway 9

ACCEPTED MANUSCRIPT components or by its upstream protein in many malignancies including PDAC.

Ma et al. (2014) established that miR-212 is a potential pro-oncogenic miRNA in PDAC which is down-regulated in colorectal cancer (Meng et al., 2013), gastric cancer (Zeng et al., 2013), hepatocellular carcinoma (Liang et al., 2013), and prostate cancer (Walter et

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al., 2013), while it is overexpressed in some other malignancies, such as oral carcinoma (Scapoli et al., 2010) and non-small cell lung cancer (Li et al., 2012) . MiR-212 is significantly overexpressed in cell lines and PDAC tissues compared with adjacent normal tissues. MiR-212 promotes PDAC cell growth and motility in vitro by targeting

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PTCH1 which is negatively correlated with miR-212 in PDAC tissues. Overexpression of tumor suppressor gene PTCH1 can reverse miR-212 induced proliferation, migration, and invasion of PDAC cell by blocking the Hh signaling pathway, indicating that miR-

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212/PTCH1 is a potential treatment target for PDAC.

Gli1 is a major transcriptional factor as well as a target gene of the hedgehog pathway, and plays an essential role in division, apoptosis, adhesion, movement, invasion and metastasis of a number of human malignancies, including PDAC cells. Inhibition of Gli1

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can suppress Shh signaling. Previous research has focused on naturally existing miRNAs. Tsuda et al. (2006) designed and synthesized an artificial miRNA-3548 that targets Gli1, and demonstrated that miRNA-3548 significantly inhibits proliferation of MiaPaCa-2 cells by delayed cell division and inducing late apoptosis. This demonstrates that

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synthetic miRNA directed towards key cell signaling components may be an effective

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treatment for PDAC (Fig. 3).

WNT SIGNALING

Wingless/integrase-1 (Wnt) signaling is a central pathway mediating various key cellular processes of embryonic development, such as proliferation, differentiation, and apoptosis (Peifer et al., 2000). However, aberrant activation of Wnt is associated with several human malignancies and appears to be one of the major drivers for pancreatic carcinogenesis (Moon et al., 2002). Nineteen mammalian Wnt ligands have been identified (Morris et al., 2010). Upon ligand binding, Frizzled family receptors trigger the activation of Wnt signalling cascades through either a canonical (β-catenindependent) or non-canonical (β-catenin-independent). 10

ACCEPTED MANUSCRIPT The canonical Wnt/β-catenin pathway is activated when Wnt binds to Frizzled/LRP receptor complexes, leading to cytoplasmic accumulation and nuclear localization of βcatenin protein. Mutations of Wnt signaling components occur in 65% of human pancreatic tumors and Wnt signaling activity is markedly increased in pancreatic

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carcinogenesis (Morris et al., 2010). β-catenin plays an essential role in regulating the transcription of Wnt target genes through Tcf/Lef family transcriptional factors. βcatenin is a transcriptional co-activator, mediating various aspects of cellular processes such as regulating cell cycle (Wei et al., 2010), promoting proliferation, stimulating

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angiogenesis (Zhang et al., 2007, 2010), inhibiting apoptosis (Ristorcelli et al., 2008; Arlt et al., 2013), inducing drug resistance (Olive et al., 2009), and maintaining cancer stem

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cells (CSCs) in PDAC (Li et al., 2007; Prakash et al., 2007).

The proto-oncogene forkhead box M1 (FOXM1) is a critical transcription factor that mediates various aspects of tumor biology and its overexpression is correlated with a poor prognosis of PDAC. FOXM1 binds to β-catenin and mediates its nuclear translocation, leading to Wnt signaling activation (Kong et al., 2013). Li et al. (2014)

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established that miR-494 functions as a negative regulator of FOXM1 by directly targeting its 3’-UTR in PDAC cells. MiR-494 suppresses cell proliferation, migration, and metastasis in vitro and in vivo by regulating the FOXM1/β-catenin signaling axis. They also established that down-regulation of miR-494 as well as overexpression of FOXM1

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affects TGF-β signaling. Down-regulation of Smad4 in PDAC tissues is critical for suppressing miR-494, leading to FOXM1 overexpression and activation of Wnt/β-

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catenin pathway, and ultimately promoting the PDAC progression.

The canonical Wnt/β-catenin signaling is also involved in chemotherapy-resistance of PDAC. Although gemcitabine, an inhibitor for DNA synthesis and a ribonucleotide reductase, has become the standard treatment for PDAC, the response rate to gemcitabine is still <20% (Oettle et al., 2007; Chan et al., 2014). Nagano et al. (2013) revealed that miR-29a induces resistance to gemcitabine through activation of Wnt/βcatenin signaling in pancreatic cancer cell lines. Previous researches also demonstrated that miR-29a potentiates the Wnt/β-catenin signaling through directly targeting the signaling antagonists, secreted frizzled related protein 2 (sFRP2), Dikkopf-1 (Dkk1), and 11

ACCEPTED MANUSCRIPT Kremen2 (Kapinas et al., 2010). The canonical Wnt signaling also induces miR-29a transcription to form a regulatory circuit. Whether such a feeback mechanism exists in gemcitabine resistant PDAC cells remains to be investigated.

Early metastasis and recurrence are the two main characteristics of PDAC, and they are

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normally attributed to the stemness of cancer cells (Brabletz et al., 2005; Mueller et al., 2009). However, the potential molecular mechanisms underlying the stem cell-like properties of PDAC cells remain largely elusive. In previous study, miRNAs were demonstrated to influence cancer cell stemness by modulating specific pathways,

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including Wnt/β-catenin signaling (Croce et al., 2005; Reya et al., 2005). MiR-29c level is significantly down-regulated in PDAC following negative regulation by TGF-β/SMAD3 signaling and is correlated with patient prognoses. Conversely, restoring miR-29c

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suppresses PDAC cell migration and invasion, which is associated with reduced stem cell-like phenotype in vitro. In addition, in vivo tumorigenicity and invasion capacity is also attenuated. MiR-29c exerts its functions by directly targeting the 3’-UTR of FRAT2, LRP6, FZD4 and FZD5, which all are the upstream regulatory genes of Wnt/β-catenin pathway. Further supporting the physiological relevance, activation of TGF-

2015).

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β/SMAD3/miR-29c/Wnt axis has been found in clinical PDAC specimens (Jiang et al.,

EMT plays an essential role in metastasis of PDAC, while expression of the adhesion

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molecule E-cadherin is a central node for cell dissemination. β-catenin participates in this main intercellular adhesion mechanism by interacting with E-cadherin to form a

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cadherin/catenin complex. Up-regulation of miR-23a and/or miR-24 could inhibit expression of FZD5, TMEM92 and/or HNF1B, leading to degradation and destabilization of the cadherin/catenin complex as well as expression of Wnt-related genes. Thus, deregulation of this complex can facilitate EMT during PDAC progression (Listing et al., 2015) (Fig. 4).

CONTROL OF CELL CYCLE Mitotic cell cycle progression is a tightly controlled sequence of four phases, G1, S, G2, and M phases, in normal cells. In cancer cells, cell cycle is significantly deregulated, which is associated with up-regulated and uncontrolled DNA replication and cellular 12

ACCEPTED MANUSCRIPT proliferation (Musgrove et al., 2011). Cyclin dependent kinases (CDKs), which are tightly governed by cyclins and CDK inhibitors (CDKIs), are critical regulatory components for G1/S phase transition (DNA replication) and G2/M phase transition (mitosis). Tumorassociated cell cycle defects are often attributed to alterations in CDK expression or/and

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activity (Malumbres et al., 2009).

Wnt signaling is an essential pathway in managing cell division by up-regulating Cyclin D, c-Myc, COX-2, peroxisome proliferator-activated receptors (PPARs), growth factors and their receptors, and down-regulating E-cadherin and cell cycle inhibitor p53. Cyclin

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D overexpression is linked to CDK4 or CDK6 activity, which are also critical for G1 checkpoint control in many malignancies. CDK4 or CDK6 inhibits the tumor suppressor protein retinoblastoma (Rb), and promotes the transition of cell cycle from G0 to G1

progression of G1/S phase transition.

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phase (Sun et al., 2007). Furthermore, Cyclin E can complex with CDK2 and promote the

Several miRNAs are involved in the deregulation of the cell cycle genes that are critical for pancreatic oncogenesis. Lodygin et al. (2008) found that miR-34a functions as a

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tumor suppressor that can induce G1 cell cycle arrest, senescence and apoptosis of cancer cells. However, in PDAC, miR-34a expression is typically down-regulated or lost, partially through epigenetic silencing, which is attributed to the CpG methylation in its promoter. However, restoration of miR-34a expression in PDAC cells can induce

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senescence and cell cycle arrest by targeting CDK6. Lee et al. (2009) revealed that miR107, which exerts its function by targeting CDK6, is also suppressed by CpG methylation

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at its promoter. MiR-183 is another miRNA that is down-regulated in PDAC and significantly associated with tumor grade, metastasis and TNM (tumor node metastasis) stage. Mir-183 regulates Cyclin D1, CDK2 and CDK4 by targeting B-cell-specific Moloney murine leukemia virus insertion site 1 (Bmi-1) (Zhou L et al., 2014).

MiR-210 targets e2f transcription factor 3 (E2F3), a cell cycle regulator, to suppress cell division, which is associated with a better prognosis of PDAC patients (Chen et al., 2012). Greither et al. (2010) established that miR-155 has a strong effect on survival of PDAC patients and functions as a biomarker for diagnosis. MiR-155 modulates LDOC1, a regulator of apoptosis, and BACH-1, a known transcriptional repressor, by directly 13

ACCEPTED MANUSCRIPT targeting their 3’-UTR to regulate the cell cycle and promote pancreatic tumorigenesis. Another important cell cycle regulator, p53, can also be regulated by miRNAs, specifically miR-203. Besides that, miR-222 can facilitate cancer cells to transit from G1 to S phase through targeting p27 (Kip1) and p57 (Kip2) (Greither et al., 2010). Ivanovska et al. (2008) found that miR-106b family members are overexpressed in

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PDAC and function to promote cell proliferation by targeting p21/CDKN1A.

Zhao et al. (2013) revealed that down-regulated miR-148b is significantly correlated with clinical characteristics, such as increased tumor size, late TNM stage, lymphatic

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invasion, and distant metastasis, indicating a worse prognosis of patients with PDAC. MiR-148b could induce cell-cycle arrest and apoptosis, enhance chemosensitivity, and inhibit cell proliferation and invasion of pancreatic cancer cells in vitro. Furthermore, it

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could suppress tumorigenicity in vivo. AMP activated protein kinase a1 (AMPKa1), which functioned as a critical promoter in tumorigenesis and overexpressed in human pancreatic cancer cells and tissues, has been established to be the direct target of miR148b and is inversely correlated with miR-148b level in PDAC tissues (Zhao et al., 2013). In the study by Liffers et al. (2011), miR-148a is also underexpressed in PDAC. They

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found miR148a modulates tumor cell growth and colony formation by targeting CDC25B to activate CDK1/Cyclin B complex.

MiR-132/-212 are overexpressed in PDAC tissues, and are shown to function as a

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promoter of tumorigenicity by targeting Rb1 (Park et al., 2011). MiR-26a is downregulated in PDAC tissues, and is correlated with a shorter survival. MiR-26a could

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inhibit cell proliferation and decrease tumor growth by binding Cyclin E2 to cause cell cycle arrest (Deng et al., 2013). MiR-219-1-3p expression is significantly downregulated in PDAC cell lines and tissues and suppresses MUC4 by directly binding to its 3’-UTR. MiR-219-1-3p could decrease cell proliferation by decreasing Akt and Erk pathway activation and Cyclin D1 production (Lahdaoui et al., 2015) (Fig. 5).

APOPTOSIS Surgical resection is regarded as a better curative treatment option for PDAC if diagnosed early, but only a minority of patients (<15%) are operable. Chemotherapy is an alternative therapeutic, but most of the time it is only a palliative treatment that 14

ACCEPTED MANUSCRIPT provides some improvements in quality of life (Rivera et al., 2009). The major obstacle in chemotherapy is mainly due to the profound drug resistance caused by a markedly reduced apoptotic responsiveness of PDAC cells (Wong et al., 2009). As a result, targeting the dysfunctional apoptotic pathway and resetting their apoptotic threshold

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could be a promising approach to overcome drug resistance (Wang et al., 2014).

Apoptosis is a critical physiological regulator in maintenance of normal cell turnover and tissue homeostasis as well as eliminating damaged, redundant, and harmful cells from an organism. There are two alternative intracellular routes of apoptosis: the

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intrinsic pathway (mitochondrial pathway) and the extrinsic pathway (death-receptor pathway) in mammals. Apoptosis also mediates an essential anti-tumor function by preventing tumorigenesis of normal cells and eliminating cancer cells. However, a

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number of strategies to evade apoptosis are utilized by malignant cells, especially in PDAC (Fulda, 2010).

B-cell lymphoma (Bcl) family, which includes anti-apoptotic proteins such as Bcl-2 and Bcl-XL, pro-apoptotic proteins such as BAX and BAK, and BH3-only containing

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subfamily, such as BID and BIM with opposing biologic functions, forms a complex network of interactions to tightly regulate the intrinsic apoptotic response (Youle et al., 2008; Kang et al., 2009). This equilibrium could be perturbed by suppressing proapoptotic proteins or overexpressing anti-apoptotic proteins, or a combination of both

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to facilitate tumor formation and progression in PDAC. The anti-apoptotic Bcl-2 family proteins are commonly overexpressed in cancer cells to overcome stress signals, and are

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correlated with chemotherapeutic resistance, recurrence, and poor prognosis. Several miRNAs have been established to be involved in the Bcl-2 family proteins mediated apoptotic pathways. MiR-181b is demonstrated to promote gemcitabine sensitivity and apoptosis of PDAC cells by directly targeting Bcl-2 (Cai et al., 2013). Conversely, miR-21 binding to the Bcl-2 3’-UTR leads to overexpression of Bcl-2, and consequently inhibits apoptosis and mediates chemosensitivity to gemcitabine in MIA PaCa-2 cells (Dong et al., 2011). In addition to affecting cancer cells, miRNAs can also mediate apoptosis of pancreatic stellate cells (PSCs) by targeting Bcl-2. In the study of Shen et al. (2012), Bcl2 protein levels were shown to be increased during PSC activation, which inversely correlated to the expression levels of miR-15b and miR-16. Restoration of miR-15b and 15

ACCEPTED MANUSCRIPT miR-16 induces apoptosis in activated PSCs by down-regulating Bcl-2 protein. Furthermore, Zhang et al. (2014) demonstrated that miR-148a acts as a tumor suppressor to inhibit cell proliferation and promote apoptosis in pancreatic cancer by targeting cholecystokinin-B receptor (CCKBR) and Bcl-2. The study by Guo et al. (2012) revealed that miR491-5p can inhibit cell proliferation and promote cell apoptosis in

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pancreatic cancer cells by suppressing Bcl-XL gene expressions to activate mitochondrial apoptotic pathway. Moreover, miR491-5p could inhibit both STAT3 and PI3K/Akt signaling pathways. MiR-15a-3p also has pro-apoptotic functions by binding to Bcl-XL 3’-UTR (Druz et al., 2013). MiR-17-5p is overexpressed in pancreatic cancer

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and plays a critical role in tumorigenesis. Inhibition of miR-17-5p can enhance cell chemosensitivity to gemcitabine, and induce apoptosis by regulating Bim expression (Yan et al., 2012). Mir-301a is also upregulated in pancreatic cancer to promote cell

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proliferation and suppresses cell apoptosis by repressing Bim (Chen et al., 2012). Data also demonstrate that some miRNAs can target BAX, which acts as an apoptosispromoter in several cancers. For example, Hamada et al. (2014) demonstrated that miR365 can induce gemcitabine resistance by targeting Src Homology 2 Domain Containing

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1 (SHC1) and BAX to increases pancreatic cancer cell survival.

Various signaling pathways have been implicated in the regulation of apoptotic machinery in different cancer types. One of the most important pathways that confer resistance to apoptosis is the Akt pathway (Franke, 2008). Several important factors,

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such as hormones, cytokines and growth factors, utilize the Akt pathway for inducing anti-apoptotic properties of cancer cells. Increased AKT activity is commonly exhibited

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in most PDAC tissues and cell lines (Bleeker et al., 2008). Song et al. (2014) revealed that miR-375 inhibits cell proliferation, arrests the cell cycle at G0/G1 and induces cell apoptosis by directly targeting 3-phosphoinositide-dependent protein kinase 1 (PDPK1) in pancreatic cancer. MiR-375 inhibits the malignant phenotype through Akt signaling pathway (Zhou et al., 2014). Oncogenic KRAS promotes tumorigenesis of PDAC through activation of multiple downstream pathways, including PI3K/Akt pathways. MiR-96 affects pancreatic cancer cell apoptosis by directly impairing the KRAS/Akt pathway as we mentioned above. AKT2 is a major downstream effector of PI3K, and is activated in up to 60% of PDAC. AKT2 activation can promote cell proliferation and resistance to chemotherapy by inhibiting apoptosis. Furthermore, overexpression of miR-615-5p can 16

ACCEPTED MANUSCRIPT inhibit cell proliferation and induce apoptosis of PDAC cell in vitro by negatively regulating AKT2 (Sun et al., 2015). IGF-1R/AKT pathway has been identified to be involved in the regulation of multiple biological processes of cancers. MiR-630 induces growth inhibition and enhances apoptosis by targeting IGF-1R (Farhana et al., 2013). MiR-497 also can inhibit proliferation, promote gemcitabine sensitivity, and induce

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apoptosis by binding to the 3’-UTR of IGF-1R (Xu et al., 2014). Eukaryotic elongation factor 1-a (eEF1A), a high expressed G protein family member, is directly targeted by miR-663 and is strongly associated with TNM stage and node metastasis of PDAC patients. Moreover, miR-663 induces cell apoptosis and attenuates pancreatic cancer

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cell proliferation and invasiveness through Akt activation (Zang et al., 2015).

The activation of JAK2/STAT3 signaling pathway is implicated in various cell processes,

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such as proliferation, apoptosis and chemotherapy resistance in various types of cancer. Wang et al. (2014) identified JAK2 as a target gene of miR-216a. MiR-216a suppresses cell growth and facilitates cell apoptosis of pancreatic cancer cells by repressing JAK2 expression. Besides that, miR-130b is also involved in STAT3 signaling pathway by directly targeting STAT3. MiR-130b is correlated with lymphatic invasion, distant

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metastasis and late TNM stage, and functions as an independent predictor for poor prognosis of pancreatic cancer patients. In the in vitro assay, miR-130b can induce apoptosis and cell cycle arrest, and suppress cell proliferation and invasiveness in PDAC

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cells (Zhao et al., 2013).

Tumor suppressor p53 plays a pivotal role in integrating various physiological stress

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signals into multiple tumor suppressive responses, especially inducing apoptosis (Vousden et al., 2009). Somatic inactivating mutations leading to p53 gene loss of function occurs in 50%–75% of PDAC. This p53 loss of function causes impaired activity of Cyclin D1 and CDK-2 and leads to uncontrolled cell proliferation and inhibition of apoptosis. Several miRNAs act as important components of the p53 transcriptional network (Morton et al., 2010). Tumor protein 53-induced nuclear protein 1 (TP53INP1) is a pro-apoptotic stress-induced p53 target gene. TP53INP1 interacts with p53 and modulates its transcriptional activity. Moreover, TP53INP1 can induce cell cycle arrest and apoptosis in several cell lines in a p53-independent manner but its expression is markedly reduced or completely lost in early stage of PDAC tumorigenesis. MiR-155 is 17

ACCEPTED MANUSCRIPT markedly up-regulated in PDAC and plays an oncogenic role by directly targeting TP53INP1 mRNA 3’UTR (Gironella et al., 2007). The inhibitor of growth (ING) family proteins consist of five isoforms and act as tumor suppressors that affect DNA repair, inducing cell cycle arrest, cellular senescence and apoptosis. ING interacts with TP53. ING can induce p53-dependent apoptosis in response to DNA damage, cause cell cycle

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arrest at G1 phase to prevent proliferation and colony formation of cancer cells. In line with that, miR-371-5p promotes cell proliferation and tumor growth by inhibiting ING1 (He et al., 2014). Overexpression of miR-214 decreased gemcitabine sensitivity of PDAC by directly binding ING4 (Zhang et al., 2010). Meanwhile, miR-196a promotes

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proliferation and invasion and inhibits apoptosis in PDAC cells by targeting ING5 3’-UTR (Liu et al., 2013).

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The nuclear factor κB (NF-κB) transcription factors are composed of five DNA binding members of the Rel family including NF-κB1 (p50/p105), NF-κB2 (p52/p100), RelA (p65), RelB, and C-Rel. NF-κB is ubiquitously expressed and is activated in eukaryotic cell systems in response to a wide range of stimuli including inflammation, bacterial and viral products, and various types of stress. Many NF-κB downstream genes are involved

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in multiple biological processes, including apoptosis, survival, cell adhesion, cellular stress responses, inflammation, and immune regulation. NF-κB is constitutively activated in most human PDAC, and plays a crucial role in linking to tumor promotion and progression, as well as chemotherapy and radiotherapy resistance. It is also

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involved in the apoptosis signaling pathway of PDAC. Lu et al. (2011) demonstrated that miR-301a is specifically up-regulated in PDAC and functions as a NF-κB activator by

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directly targeting NF-κB-repressing factor (NKRF). The persistently activated NF-kB could further promote miR-301a transcription, which forms a positive feedback loop. Moreover, the inhibition of miR-301a in PDAC cells could suppress NF-κB downstream gene expression and attenuates tumor growth in vivo. Takiuchi et al. (2013) revealed that expression of miR-181b is correlated with chemoresistance to gemcitabine. MiR181b inhibits the response to gemcitabine and suppresses apoptosis by directly targeting cylindromatosis (CYLD) protein, which acts as a tumor suppressor gene and negatively regulates NF-κB activity. In the research by Huang et al. (2014), miR-196a was found to be overexpressed in human PDAC cell lines. MiR196a enhances cell proliferation and migration through binding to the 3’-UTR of NF-κB-inhibitor α 18

ACCEPTED MANUSCRIPT (NFKBIA). Moreover, inhibition of miR-196a in PDAC cells suppresses their proliferation and migration with an increase in G0/G1 transition and down-regulates Cyclin D1 and CDK4/6 (Fig. 6).

CONCLUSION

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Deregulated miRNAs play pivotal roles in PDAC development and progression by affecting multiple cellular processes including cell proliferation, apoptosis, survival, invasion, metastasis, and chemotherapeutic resistance of PDAC. By broadening our understanding of the molecular mechanisms between miRNAs and pancreatic

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oncogenesis, miRNAs can provide new avenues for the development of novel targeted therapeutic strategies. MiRNA expression profiles should be investigated to evaluate the potential function as biomarkers for early diagnosis, disease progression, response to

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therapy, and prognosis because of their characteristics of high stability, tissue specificity and ease of availability.

ACKNOWLEDGMENT

Longhao Sun is supported by a fellowship from Tianjin Medical University General

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Hospital for his postdoctoral fellow research at MD Anderson Cancer Center.

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ACCEPTED MANUSCRIPT REFERENCES

AC C

EP

TE D

M AN U

SC

RI PT

Arlt, A., Müerköster, S.S., Schäfer, H., 2013. Targeting apoptosis pathways in pancreatic cancer. Cancer Lett. 332, 346-358. Bai, S., Cao, X., 2002. A nuclear antagonistic mechanism of inhibitory Smads in transforming growth factor-beta signaling. J. Biol. Chem. 277, 4176-4182. Bleeker, F.E., Felicioni, L., Buttitta, F., Lamba, S., Cardone, L., Rodolfo, M., Scarpa, A., Leenstra, S., Frattini, M., Barbareschi, M., Grammastro, M.D., Sciarrotta, M.G., Zanon, C., Marchetti, A., Bardelli, A., 2008. AKT1 (E17K) in human solid tumours. Oncogene 27, 5648-5650. Brabletz, T., Jung, A., Spaderna, S., Hlubek, F., Kirchner, T., 2005. Opinion: migrating cancer stem cells-an integrated concept of malignant tumour progression. Nat. Rev. Cancer 5, 744-749. Broderick, J.A., Zamore, P.D., 2011. MicroRNA therapeutics. Gene Ther. 18, 1104-1110. Burk, U., Schubert, J., Wellner, U., Schmalhofer, O., Vincan, E., Spaderna, S., Brabletz, T., 2008. A reciprocal repression between ZEB1 and members of the miR-200 family promotes EMT and invasion in cancer cells. EMBO Rep. 9, 582-589. Cai, B., An, Y., Lv, N., Chen, J., Tu, M., Sun, J., Wu, P., Wei, J., Jiang, K., Miao, Y., 2013. miRNA-181b increases the sensitivity of pancreatic ductal adenocarcinoma cells to gemcitabine in vitro and in nude mice by targeting BCL-2. Oncol. Rep. 29, 1769-1776. Chan, S.L., Chan, S.T., Chan, E.H., He, Z.X., 2014. Systemic treatment for inoperable pancreatic adenocarcinoma: review and update. Chin. J. Cancer 33, 267-276. Chen, W.Y., Liu, W.J., Zhao, Y.P., Zhou, L., Zhang, T.P., Chen, G., Shu, H., 2012. Induction, modulation and potential targets of miR-210 in pancreatic cancer cells. Hepatobiliary Pancreat. Dis. Int. 11, 319-324. Chen, Z., Chen, L.Y., Dai, H.Y., Wang, P., Gao, S., Wang, K., 2012. miR-301a promotes pancreatic cancer cell proliferation by directly inhibiting Bim expression. J. Cell. Biochem. 113, 32293235. Croce, C.M., Calin, G.A., 2005. miRNAs, cancer, and stem cell division. Cell 122, 6-7. Croce, C.M., 2009. Causes and consequences of microRNA dysregulation in cancer. Nat. Rev. Genet. 10, 704-714. Deng, J., He, M., Chen, L., Chen, C., Zheng, J., Cai, Z., 2013. The loss of miR-26a-mediated posttranscriptional regulation of cyclin E2 in pancreatic cancer cell proliferation and decreased patient survival. PLoS One 8, e76450. Ding, X.M., 2014. MicroRNAs: regulators of cancer metastasis and epithelial-mesenchymal transition (EMT). Chin. J. Cancer 33, 140-147. Dong, J., Zhao, Y.P., Zhou, L., Zhang, T.P., Chen, G., 2011. Bcl-2 upregulation induced by miR-21 via a direct interaction is associated with apoptosis and chemoresistance in MIA PaCa-2 pancreatic cancer cells. Arch. Med. Res. 42, 8-14. Dosch, J.S., Pasca di Magliano, M., Simeone, D.M., 2010. Pancreatic cancer and hedgehog pathway signaling: new insights. Pancreatology 10, 151-157. Druz, A., Chen, Y.C., Guha, R., Betenbaugh, M., Martin, S.E., Shiloach, J., 2013. Large-scale screening identifies a novel microRNA, miR-15a-3p, which induces apoptosis in human cancer cell lines. RNA Biol. 10, 287-300. Farhana, L., Dawson, M.I., Murshed, F., Das, J.K., Rishi, A.K., Fontana, J.A., 2013. Upregulation of miR-150* and miR-630 induces apoptosis in pancreatic cancer cells by targeting IGF-1R. PLoS One 8, e61015. Frampton, A.E., Krell, J., Jacob, J., Stebbing, J., Castellano, L., Jiao, L.R., 2012. Loss of miR-126 is crucial to pancreatic cancer progression. Expert Rev. Anticancer Ther. 7, 881-884. 20

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Franke, T.F., 2008. PI3K/Akt: getting it right matters. Oncogene 27, 6473-6488. Fulda, S., 2010. Evasion of apoptosis as a cellular stress response in cancer. Int. J. Cell Biol. 2010, 370835. Gironella, M., Seux, M., Xie, M.J., Cano, C., Tomasini, R., Gommeaux, J., Garcia, S., Nowak, J., Yeung, M.L., Jeang, K.T., Chaix, A., Fazli, L., Motoo, Y., Wang, Q., Rocchi, P., Russo, A., Gleave, M., Dagorn, J.C., Iovanna, J.L., Carrier, A., Pébusque, M.J., Dusetti, N.J., 2007. Tumor protein 53induced nuclear protein 1 expression is repressed by miR-155, and its restoration inhibits pancreatic tumor development. Proc. Natl. Acad. Sci. USA 104, 16170-16175. Gregory, P.A., Bert, A.G., Paterson, E.L., Barry, S.C., Tsykin, A., Farshid, G., Vadas, M.A., KhewGoodall, Y., Goodall, G.J., 2008. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat. Cell Biol. 10, 593-601. Greither, T., Grochola, L.F., Udelnow, A., Lautenschläger, C., Würl, P., Taubert, H., 2010. Elevated expression of microRNAs 155, 203, 210 and 222 in pancreatic tumors is associated with poorer survival. Int. J. Cancer 126, 73-80. Guo, R., Wang, Y., Shi, W.Y., Liu, B., Hou, S.Q., Liu, L., 2012. MicroRNA miR-491-5p targeting both TP53 and Bcl-XL induces cell apoptosis in SW1990 pancreatic cancer cells through mitochondria mediated pathway. Molecules 17, 14733-14747. Hamada, S., Satoh, K., Fujibuchi, W., Hirota, M., Kanno, A., Unno, J., Masamune, A., Kikuta, K., Kume, K., Shimosegawa, T., 2012. MiR-126 acts as a tumor suppressor in pancreatic cancer cells via the regulation of ADAM9. Mol. Cancer Res. 10, 3-10. Hamada, S., Masamune, A., Miura, S., Satoh, K., Shimosegawa, T., 2014. MiR-365 induces gemcitabine resistance in pancreatic cancer cells by targeting the adaptor protein SHC1 and pro-apoptotic regulator BAX. Cell Signal. 26, 179-185. Hao, J., Zhang, S., Zhou, Y., Hu, X., Shao, C., 2011. MicroRNA 483-3p suppresses the expression of DPC4/Smad4 in pancreatic cancer. FEBS Lett. 585, 207-213. Hao, J., Zhang, S., Zhou, Y., Liu, C., Hu, X., Shao, C., 2011. MicroRNA 421 suppresses DPC4/Smad4 in pancreatic cancer. Biochem. Biophys. Res. Commun. 406, 552-557. Harazono, Y., Muramatsu, T., Endo, H., Uzawa, N., Kawano, T., Harada, K., Inazawa, J., Kozaki, K., 2013. miR-655 is an EMT-suppressive microRNA targeting ZEB1 and TGFBR2. PLoS One 8, e62757. Hata, A., Davis, B.N., 2009. Control of microRNA biogenesis by TGFbeta signaling pathway-A novel role of Smads in the nucleus. Cytokine Growth Factor Rev. 20, 517-521. He, D., Miao, H., Xu, Y., Xiong, L., Wang, Y., Xiang, H., Zhang, H., Zhang, Z., 2014. MiR-371-5p facilitates pancreatic cancer cell proliferation and decreases patient survival. PLoS One 9, e112930. Hidalgo, M., 2010. Pancreatic cancer. N. Engl. J. Med. 362, 1605-1617. Hu, Y., Ou, Y., Wu, K., Chen, Y., Sun, W., 2012. MiR-143 inhibits the metastasis of pancreatic cancer and an associated signaling pathway. Tumour Biol. 33, 1863-1870. Huang, F., Tang, J., Zhuang, X., Zhuang, Y., Cheng, W., Chen, W., Yao, H., Zhang, S., 2014. MiR-196a promotes pancreatic cancer progression by targeting nuclear factor kappa-B-inhibitor alpha. PLoS One 9, e87897. Huang, T., Alvarez, A., Hu, B., Cheng, S.Y., 2013. Noncoding RNAs in cancer and cancer stem cells. Chin. J. Cancer 32, 582-593. Hui, C.C., Angers, S., 2011. Gli proteins in development and disease. Annu. Rev. Cell Dev. Biol. 27, 513-537. Ivanovska, I., Ball, A.S., Diaz, R.L., Magnus, J.F., Kibukawa, M., Schelter, J.M., Kobayashi, S.V., Lim, 21

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

L., Burchard, J., Jackson, A.L., Linsley, P.S., Cleary, M.A., 2008. MicroRNAs in the miR-106b family regulate p21/CDKN1A and promote cell cycle progression. Mol. Cell. Biol. 28, 21672174. Izumchenko, E., Chang, X., Michailidi, C., Kagohara, L., Ravi, R., Paz, K., Brait, M., Hoque, M.O., Ling, S., Bedi, A., Sidransky, D., 2014. The TGFβ-miR200-MIG6 pathway orchestrates the EMTassociated kinase switch that induces resistance to EGFR inhibitors. Cancer Res. 74, 39954005. Jiang, J., Yu, C., Chen, M., Zhang, H., Tian, S., Sun, C., 2015. Reduction of miR-29c enhances pancreatic cancer cell migration and stem cell-like phenotype. Oncotarget 6, 2767-2778. Jiao, L.R., Frampton, A.E., Jacob, J., Pellegrino, L., Krell, J., Giamas, G., Tsim, N., Vlavianos, P., Cohen, P., Ahmad, R., Keller, A., Habib, N.A., Stebbing, J., Castellano, L., 2012. MicroRNAs targeting oncogenes are down-regulated in pancreatic malignant transformation from benign tumors. PLoS One 7, e32068. Jones, S., Zhang, X., Parsons, D.W., Lin, J.C., Leary, R.J., Angenendt, P., Mankoo, P., Carter, H., Kamiyama, H., Jimeno, A., Hong, S.M., Fu, B., Lin, M.T., Calhoun, E.S., Kamiyama, M., Walter, K., Nikolskaya, T., Nikolsky, Y., Hartigan, J., Smith, D.R., Hidalgo, M., Leach, S.D., Klein, A.P., Jaffee, E.M., Goggins, M., Maitra, A., Iacobuzio-Donahue, C., Eshleman, J.R., Kern, S.E., Hruban, R.H., Karchin, R., Papadopoulos, N., Parmigiani, G., Vogelstein, B., Velculescu, V.E., Kinzler, K.W., 2008. Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science 321, 1801-1806. Vousden, K.H., Prives, C., 2009. Blinded by the light: the growing complexity of p53. Cell 137, 413-431. Kapinas, K., Kessler, C., Ricks, T., Gronowicz, G., Delany, A.M., 2010. miR-29 modulates Wnt signaling in human osteoblasts through a positive feedback loop. J. Biol. Chem. 285, 2522125231. Kang, M.H., Reynolds, C.P., 2009. Bcl-2 inhibitors: targeting mitochondrial apoptotic pathways in cancer therapy. Clin. Cancer Res. 15, 1126-1132. Keklikoglou, I., Hosaka, K., Bender, C., Bott, A., Koerner, C., Mitra, D., Will, R., Woerner, A., Muenstermann, E., Wilhelm, H., Cao, Y., Wiemann, S., 2014. MicroRNA-206 functions as a pleiotropic modulator of cell proliferation, invasion and lymphangiogenesis in pancreatic adenocarcinoma by targeting ANXA2 and KRAS genes. Oncogene 1-12. Kent, O.A., Chivukula, R.R., Mullendore, M., Wentzel, E.A., Feldmann, G., Lee, K.H., Liu, S., Leach, S.D., Maitra, A., Mendell, J.T., 2010. Repression of the miR-143/145 cluster by oncogenic Ras initiates a tumor-promoting feed-forward pathway. Genes Dev. 24, 2754-2759. Kong, X., Li, L., Li, Z., Le, X., Huang, C., Jia, Z., Cui, J., Huang, S., Wang, L., Xie, K., 2013. Dysregulated expression of FOXM1 isoforms drives progression of pancreatic cancer. Cancer Res. 73, 3987-3996. Lahdaoui, F., Delpu, Y., Vincent, A., Renaud, F., Messager, M., Duchêne, B., Leteurtre, E., Mariette, C., Torrisani, J., Jonckheere, N., Van Seuningen, I., 2015. miR-219-1-3p is a negative regulator of the mucin MUC4 expression and is a tumor suppressor in pancreatic cancer. Oncogene 34, 780-788. Lee, K.H., Lotterman, C., Karikari, C., Omura, N., Feldmann, G., Habbe, N., Goggins, M.G., Mendell, J.T., Maitra, A., 2009. Epigenetic silencing of MicroRNA miR-107 regulates cyclin-dependent kinase 6 expression in pancreatic cancer. Pancreatology 9, 293-301. Li, C., Heidt, D.G., Dalerba, P., Burant, C.F., Zhang, L., Adsay, V., Wicha, M., Clarke, M.F., Simeone, D.M., 2007. Identification of pancreatic cancer stem cells. Cancer Res. 67, 1030-1037. 22

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Li, L., Li, Z., Kong, X., Xie, D., Jia, Z., Jiang, W., Cui, J., Du, Y., Wei, D., Huang, S., Xie, K., 2014. Downregulation of microRNA-494 via loss of SMAD4 increases FOXM1 and β-catenin signaling in pancreatic ductal adenocarcinoma cells. Gastroenterology 147, 485-497. Li, Y., Zhang, D., Chen, C., Ruan, Z., Li, Y., Huang, Y., 2012. MicroRNA-212 displays tumorpromoting properties in non-small cell lung cancer cells and targets the hedgehog pathway receptor PTCH1. Mol. Biol. Cell 23, 1423-1434. Liang, X., Zeng, J., Wang, L., Fang, M., Wang, Q., Zhao, M., Xu, X., Liu, Z., Li, W., Liu, S., Yu, H., Jia, J., Chen, C., 2013. Histone demethylase retinoblastoma binding protein 2 is overexpressed in hepatocellular carcinoma and negatively regulated by hsa-miR-212. PLoS One 8, e69784. Liffers, S.T., Munding, J.B., Vogt, M., Kuhlmann, J.D., Verdoodt, B., Nambiar, S., Maghnouj, A., Mirmohammadsadegh, A., Hahn, S.A., Tannapfel, A., 2011. MicroRNA-148a is downregulated in human pancreatic ductal adenocarcinomas and regulates cell survival by targeting CDC25B. Lab. Invest. 91, 1472-1479. Listing, H., Mardin, W.A., Wohlfromm, S., Mees, S.T., Haier, J., 2015. MiR-23a/-24-induced gene silencing results in mesothelial cell integration of pancreatic cancer. Br. J. Cancer 112, 131139. Liu, M., Du, Y., Gao, J., Liu, J., Kong, X., Gong, Y,, Li, Z., Wu, H., Chen, H., 2013. Aberrant expression miR-196a is associated with abnormal apoptosis, invasion, and proliferation of pancreatic cancer cells. Pancreas 42, 1169-1181. Liu, M., Zhang, X., Hu, C.F., Xu, Q., Zhu, H.X., Xu, N.Z., 2014. MicroRNA-mRNA functional pairs for cisplatin resistance in ovarian cancer cells. Chin. J. Cancer 33, 285-294. Lodygin, D., Tarasov, V., Epanchintsev, A., Berking, C., Knyazeva, T., Körner, H., Knyazev, P., Diebold, J., Hermeking, H., 2008. Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer. Cell Cycle 7, 2591-2600. Lu, Z., Li, Y., Takwi, A., Li, B., Zhang, J., Conklin, D.J., Young, K.H., Martin, R., Li, Y., 2011. miR-301a as an NF-κB activator in pancreatic cancer cells. EMBO J. 30, 57-67. di Magliano, M.P., Logsdon, C.D., 2013. Roles for KRAS in pancreatic tumor development and progression. Gastroenterology 144, 1220-1229. Ma, C., Nong, K., Wu, B., Dong, B., Bai, Y., Zhu, H., Wang, W., Huang, X., Yuan, Z., Ai, K., 2014. miR212 promotes pancreatic cancer cell growth and invasion by targeting the hedgehog signaling pathway receptor patched-1. J. Exp. Clin. Cancer Res. 33, 54. Malumbres, M., Barbacid, M., 2009. Cell cycle, CDKs and cancer: a changing paradigm. Nat. Rev. Cancer 9, 153-166. Meng, X., Wu, J., Pan, C., Wang, H., Ying, X., Zhou, Y., Yu, H., Zuo, Y., Pan, Z., Liu, R.Y., Huang, W., 2013. Genetic and epigenetic down-regulation of microRNA-212 promotes colorectal tumor metastasis via dysregulation of MnSOD. Gastroenterology 145, 426-436. Moon, R.T., Bowerman, B., Boutros, M., Perrimon, N., 2002. The promise and perils of Wnt signaling through beta-catenin. Science 296, 1644 -1646. Morris, J.P.4th., Cano, D.A., Sekine, S., Wang, S.C., Hebrok, M., 2010. Beta-catenin blocks Krasdependent reprogramming of acini into pancreatic cancer precursor lesions in mice. J. Clin. Invest. 120, 508-520. Morris, J,P.4th., Wang, S.C., Hebrok, M., 2010. KRAS, Hedgehog, Wnt and the twisted developmental biology of pancreatic ductal adenocarcinoma. Nat. Rev. Cancer 10, 683-695. Morton, J.P., Timpson, P., Karim, S.A., Ridgway, R.A., Athineos, D., Doyle, B., Jamieson, N.B., Oien, K.A., Lowy, A.M., Brunton, V.G., Frame, M.C., Evans, T.R., Sansom, O.J., 2010. Mutant p53 drives metastasis and overcomes growth arrest/senescence in pancreatic cancer. Proc. Natl. 23

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Acad. Sci. USA 107, 246-251. Mu, Y., Gudey, S.K., Landström, M, 2012. Non-Smad signaling pathways. Cell Tissue Res. 347, 1120. Mueller, M.T., Hermann, P.C., Witthauer, J., Rubio-Viqueira, B., Leicht, S.F., Huber, S., Ellwart, J.W., Mustafa, M., Bartenstein, P., D’Haese, J.G., Schoenberg, M.H., Berger, F., Jauch, K.W., Hidalgo, M., Heeschen, C., 2009. Combined targeted treatment to eliminate tumorigenic cancer stem cells in human pancreatic cancer. Gastroenterology 137, 1102-1113. Musgrove, E.A., Caldon, C.E., Barraclough, J., Stone, A., Sutherland, R.L., 2011. Cyclin D as a therapeutic target in cancer. Nat. Rev. Cancer 11, 558-72. Nagano, H., Tomimaru, Y., Eguchi, H., Hama, N., Wada, H., Kawamoto, K., Kobayashi, S., Mori, M., Doki, Y., 2013. MicroRNA-29a induces resistance to gemcitabine through the Wnt/β-catenin signaling pathway in pancreatic cancer cells. Int. J. Oncol. 43, 1066-1072. Oettle, H., Post, S., Neuhaus, P., Gellert, K., Langrehr, J., Ridwelski, K., Schramm, H., Fahlke, J., Zuelke, C., Burkart, C., Gutberlet, K., Kettner, E., Schmalenberg, H., Weigang-Koehler, K., Bechstein, W.O., Niedergethmann, M., Schmidt-Wolf, I., Roll, L., Doerken, B., Riess, H., 2007. Adjuvant chemotherapy with gemcitabine vs observation in patients undergoing curativeintent resection of pancreatic cancer: a randomized controlled trial. JAMA 297, 267-277. Olive, K.P., Jacobetz, M.A., Davidson, C.J., Gopinathan, A., McIntyre, D., Honess, D., Madhu, B., Goldgraben, M.A., Caldwell, M.E., Allard, D., Frese, K.K., Denicola, G., Feig, C., Combs, C., Winter, S.P., Ireland-Zecchini, H., Reichelt, S., Howat, W.J., Chang, A., Dhara, M., Wang, L., Rückert, F., Grützmann, R., Pilarsky, C., Izeradjene, K., Hingorani, S.R., Huang, P., Davies, S.E., Plunkett, W., Egorin, M., Hruban, R.H., Whitebread, N., McGovern, K., Adams, J., IacobuzioDonahue, C., Griffiths, J., Tuveson, D.A., 2009. Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer. Science 324, 1457-1461. Ozdamar, B., Bose, R., Barrios-Rodiles ,M., Wang, H.R., Zhang, Y., Wrana, J.L., 2005. Regulation of the polarity protein Par6 by TGFbeta receptors controls epithelial cell plasticity. Science 307, 1603-1609. Padua, D., Massagué, J., 2009. Roles of TGFbeta in metastasis. Cell Res. 19, 89-102. Pan, Y., Bai, C.B., Joyner, A.L., Wang, B., 2006. Sonic hedgehog signaling regulates Gli2 transcriptional activity by suppressing its processing and degradation. Mol. Cell. Biol. 26, 3365-3377. Park, J.K., Henry, J.C., Jiang, J., Esau, C., Gusev, Y., Lerner, M.R., Postier, R.G., Brackett, D.J., Schmittgen, T.D., 2011. miR-132 and miR-212 are increased in pancreatic cancer and target the retinoblastoma tumor suppressor. Biochem. Biophys. Res. Commun. 406, 518-523. Peifer, M., Polakis, P., 2000. Wnt signaling in oncogenesis and embryogenesis-a look outside the nucleus. Science 287, 1606-1609. Prakash, N., Wurst, W., 2007. A Wnt signal regulates stem cell fate and differentiation in vivo. Neurodegener. Dis. 4, 333-338. Pylayeva-Gupta, Y., Grabocka, E., Bar-Sagi, D., 2011. RAS oncogenes: weaving a tumorigenic web. Nat. Rev. Cancer 11, 761-774. Reya, T., Clevers, H., 2005. Wnt signalling in stem cells and cancer. Nature 434, 843-850. Ristorcelli, E., Beraud, E., Verrando, P., Villard, C., Lafitte, D., Sbarra, V., Lombardo, D., Verine, A., 2008. Human tumor nanoparticles induce apoptosis of pancreatic cancer cells. FASEB J. 22, 3358-3369. Rivera, F., López-Tarruella, S., Vega-Villegas, M.E., Salcedo, M., 2009. Treatment of advanced pancreatic cancer: from gemcitabine single agent to combinations and targeted therapy. 24

ACCEPTED MANUSCRIPT

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EP

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Cancer Treat. Rev. 35, 335-339. Rosenbluh, J., Wang, X., Hahn, W.C., 2014. Genomic insights into WNT/β-catenin signaling. Trends Pharmacol. Sci. 35, 103-109. Rosty, C., Goggins, M., 2002. Early detection of pancreatic carcinoma. Hematol. Oncol. Clin. North Am. 16, 37-52. Rubin, L.L., de Sauvage, F.J., 2006. Targeting the Hedgehog pathway in cancer. Nat. Rev. Drug Discov. 5, 1026-1033. Samavarchi-Tehrani, P., Golipour, A., David, L., Sung, H.K., Beyer, T.A., Datti, A., Woltjen, K., Nagy, A., Wrana, J.L., 2010. Functional genomics reveals a BMP-driven mesenchymal-toepithelial transition in the initiation of somatic cell reprogramming. Cell Stem Cell 7, 64-77. Scapoli, L., Palmieri, A., Lo, M.L., Pezzetti, F., Rubini, C., Girardi, A., Farinella, F., Mazzotta, M., Carinci, F., 2010. MicroRNA expression profiling of oral carcinoma identifies new markers of tumor progression. Int. J. Immunopathol. Pharmacol. 23, 1229-1234. Shen, J., Wan, R., Hu, G., Yang, L., Xiong, J., Wang, F., Shen, J., He, S., Guo, X., Ni, J., Guo, C., Wang, X., 2012. miR-15b and miR-16 induce the apoptosis of rat activated pancreatic stellate cells by targeting Bcl-2 in vitro. Pancreatology 12, 91-99. Shi, W., Sun, C., He, B., Xiong, W., Shi, X., Yao, D., Cao, X., 2004. GADD34-PP1c recruited by Smad7 dephosphorylates TGFbeta type I receptor. J. Cell Biol. 164, 291-300. Siegel, R., Ma, J., Zou, Z., Jemal, A., 2014. Cancer statistics, 2014. CA. Cancer J. Clin. 64, 9-29. Singh, P., Srinivasan, R., Wig, J.D., 2011. Major molecular markers in pancreatic ductal adenocarcinoma and their roles in screening, diagnosis, prognosis, and treatment. Pancreas 40, 644-652. Singh, P., Srinivasan, R., Wig, J.D., Radotra, B.D., 2011. A study of Smad4, Smad6 and Smad7 in surgically resected samples of pancreatic ductal adenocarcinoma and their correlation with clinicopathological parameters and patient survival. BMC. Res. Notes 4, 560-564. Slack, F.J., Weidhaas, J.B., 2008. MicroRNA in cancer prognosis. N Engl J Med. 359, 2720-2722. Song, S.D., Zhou, J., Zhou, J., Zhao, H., Cen, J.N., Li, D.C., 2013. MicroRNA-375 targets the 3phosphoinositide-dependent protein kinase-1 gene in pancreatic carcinoma. Oncol Lett. 6, 953-959. Spaderna, S., Schmalhofer, O., Hlubek, F., Berx, G., Eger, A., Merkel, S., Jung, A., Kirchner, T., Brabletz, T., 2006. A transient, EMT-linked loss of basement membranes indicates metastasis and poor survival in colorectal cancer. Gastroenterology 131, 830-840. Spaderna, S., Schmalhofer, O., Wahlbuhl, M., Dimmler, A., Bauer, K., Sultan, A., Hlubek, F., Jung, A., Strand, D., Eger, A., Kirchner, T., Behrens, J., Brabletz, T., 2008. The transcriptional repressor ZEB1 promotes metastasis and loss of cell polarity in cancer. Cancer Res. 68, 537-544. Sun, A., Bagella, L., Tutton, S., Romano, G., Giordano, A., 2007. From G0 to S phase: a view of the roles played by the retinoblastoma (Rb) family members in the Rb-E2F pathway. J. Cell.Biochem. 102, 1400-1404. Sun, Y., Guo, F., Bagnoli, M., Xue, F.X., Sun, B.C., Shmulevich, I., Mezzanzanica, D., Chen, K.X., Sood, A.K., Yang, D., Zhang, W., 2015. Key nodes of a microRNA network associated with the integrated mesenchymal subtype of high-grade serous ovarian cancer. Chin. J. Cancer 34, 28-40. Sun, Y., Zhang, T., Wang, C., Jin, X., Jia, C., Yu, S., Chen, J., 2015. MiRNA-615-5p functions as a tumor suppressor in pancreatic ductal adenocarcinoma by targeting AKT2. PLoS One 10, e0119783. Takiuchi, D., Eguchi, H., Nagano, H., Iwagami, Y., Tomimaru, Y., Wada, H., Kawamoto, K., 25

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Kobayashi, S., Marubashi, S., Tanemura, M., Mori, M., Doki, Y., 2013. Involvement of microRNA-181b in the gemcitabine resistance of pancreatic cancer cells. Pancreatology 13, 517-523. Thayer, S.P., di Magliano, M.P., Heiser, P.W., Nielsen, C.M., Roberts, D.J., Lauwers, G.Y., Qi, Y.P., Gysin, S., Fernández-del Castillo, C., Yajnik, V., Antoniu, B., McMahon, M., Warshaw, A.L., Hebrok, M., 2003. Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis. Nature 425, 851-856. Tian, M., Neil, J.R., Schiemann, W.P., 2011. Transforming growth factor-β and the hallmarks of cancer. Cell. Signal. 23, 951-962. Torrisani, J., Bournet, B., du Rieu, M.C., Bouisson, M., Souque, A., Escourrou, J., Buscail, L., Cordelier, P., 2009. Let-7 MicroRNA transfer in pancreatic cancer-derived cells inhibits in vitro cell proliferation but fails to alter tumor progression. Hum. Gene Ther. 20, 831-844. Tsuda, N., Ishiyama, S., Li, Y., Ioannides, C.G., Abbruzzese, J.L., Chang, D.Z., 2006. Synthetic microRNA designed to target glioma-associated antigen 1 transcription factor inhibits division and induces late apoptosis in pancreatic tumor cells. Clin. Cancer Res. 12, 65576564. van den Brink, G.R., 2007. Hedgehog signaling in development and homeostasis of the gastrointestinal tract. Physiol. Rev. 87, 1343-1375. Walter, B.A., Valera, V.A., Pinto, P.A., Merino, M.J., 2013. Comprehensive microRNA profiling of prostate cancer. J. Cancer 4, 350-357. Wang, P., Fan, J., Chen, Z., Meng, Z.Q., Luo, J.M., Lin, J.H., Zhou, Z.H., Chen, H., Wang, K., Xu, Z.D., Liu, L.M., 2009. Low-level expression of Smad7 correlates with lymph node metastasis and poor prognosis in patients with pancreatic cancer. Ann. Surg. Oncol. 16, 826-835. Wang, R.A., Li, Z.S., Yan, Q.G., Bian, X.W., Ding, Y.Q., Du, X., Sun, B.C., Sun, Y.T., Zhang, X.H., 2014. Resistance to apoptosis should not be taken as a hallmark of cancer. Chin. J. Cancer 33, 4750. Wang, S., Chen, X., Tang, M., 2014. MicroRNA-216a inhibits pancreatic cancer by directly targeting Janus kinase 2. Oncol. Rep. 32, 2824-2830. Watanabe, S., Ueda, Y., Akaboshi, S., Hino, Y., Sekita, Y., Nakao, M., 2009. HMGA2 maintains oncogenic RAS-induced epithelial-mesenchymal transition in human pancreatic cancer cells. Am. J. Pathol. 174, 854-868. Wei, D., Wang, L., Kanai, M., Jia, Z., Le, X., Li, Q., Wang, H., Xie, K., 2010. KLF4α up-regulation promotes cell cycle progression and reduces survival time of patients with pancreatic cancer. Gastroenterology 139, 2135-2145. Wong, H.H., Lemoine, N.R., 2009. Pancreatic cancer: molecular pathogenesis and new therapeutic targets. Nat. Rev. Gastroenterol. Hepatol. 6, 412-422. Xu, J.W., Wang, T.X., You, L., Zheng, L.F., Shu, H., Zhang, T.P., Zhao, Y.P., 2014. Insulin-like growth factor 1 receptor (IGF-1R) as a target of MiR-497 and plasma IGF-1R levels associated with TNM stage of pancreatic cancer. PLoS One. 9 e92847. Yan, H.J., Liu, W.S., Sun, W.H., Wu, J., Ji, M., Wang, Q., Zheng, X., Jiang, J.T., Wu, C.P., 2012. miR-175p inhibitor enhances chemosensitivity to gemcitabine via upregulating Bim expression in pancreatic cancer cells. Dig. Dis. Sci. 57, 3160-3167. Youle, R.J., Strasser, A., 2008. The BCL-2 protein family: opposing activities that mediate cell death. Nat. Rev. Mol. Cell Biol. 9, 47-59. Yu, S., Lu, Z., Liu, C., Meng, Y., Ma, Y., Zhao, W., Liu, J., Yu, J., Chen, J., 2010. miRNA-96 suppresses KRAS and functions as a tumor suppressor gene in pancreatic cancer. Cancer Res. 70, 601526

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6025. Zang, W., Wang, Y., Wang, T., Du, Y., Chen, X., Li, M., Zhao, G., 2015. miR-663 attenuates tumor growth and invasiveness by targeting eEF1A2 in pancreatic cancer. Mol. Cancer 14, 37. Zeng, J.P., Fang, M., Wang, L.X., Liang, X.M., Shen, Y.Q., Yu, H., Liu, Z.F., Sun, Y.D., Liu, S.L., Chen, C.Y., Jia, J.H., 2013. MicroRNA-212 inhibits proliferation of gastric cancer by directly repressing retinoblastoma binding protein 2. J. Cell. Biochem. 114, 2666-2672. Zhang, B., Ma, J.X., 2010. Wnt pathway antagonists and angiogenesis. Protein Cell 1, 898-906. Zhang, J., Jia, Z., Li, Q., Wang, L., Rashid, A., Zhu, Z., Evans, D.B., Vauthey, J.N., Xie, K., Yao, J.C., 2007. Elevated expression of vascular endothelial growth factor correlates with increased angiogenesis and decreased progression-free survival among patients with low-grade neuroendocrine tumors. Cancer 109, 1478-1486. Zhang, R., Li, M., Zang, W., Chen, X., Wang, Y., Li, P., Du, Y., Zhao, G., Li, L., 2014. MiR-148a regulates the growth and apoptosis in pancreatic cancer by targeting CCKBR and Bcl-2. Tumor Biol. 35, 837-844. Zhang, X.J., Ye, H., Zeng, C.W., He, B., Zhang, H., Chen, Y.Q., 2010. Dysregulation of miR-15a and miR-214 in human pancreatic cancer. J. Hematol. Oncol. 3, 46. Zhao, G., Zhang, J.G., Shi, Y., Qin, Q., Liu, Y., Wang, B., Tian, K., Deng, S.C., Li, X., Zhu, S., Gong, Q., Niu, Y., Wang, C.Y., 2013. MiR-130b is a prognostic marker and inhibits cell proliferation and invasion in pancreatic cancer through targeting STAT3. PLoS One 8, e73803. Zhao, G., Zhang, J.G., Liu, Y., Qin, Q., Wang, B., Tian, K., Liu, L., Li, X., Niu, Y., Deng, S.C., Wang, C.Y., 2013. miR-148b functions as a tumor suppressor in pancreatic cancer by targeting AMPKα1. Mol. Cancer Ther. 12, 83-93. Zhao, W.G., Yu, S.N., Lu, Z.H., Ma, Y.H., Gu, Y.M., Chen, J., 2010. The miR-217 microRNA functions as a potential tumor suppressor in pancreatic ductal adenocarcinoma by targeting KRAS. Carcinogenesis 31, 1726-1733. Zhou, J., Song, S., He, S., Zhu, X., Zhang, Y., Yi, B., Zhang, B., Qin, G., Li, D., 2014. MicroRNA-375 targets PDK1 in pancreatic carcinoma and suppresses cell growth through the Akt signaling pathway. Int. J. Mol. Med. 33, 950-956. Zhou, L., Zhang, W.G., Wang, D.S., Tao, K.S., Song, W.J., Dou, K.F., 2014. MicroRNA-183 is involved in cell proliferation, survival and poor prognosis in pancreatic ductal adenocarcinoma by regulating Bmi-1. Oncol. Rep. 32, 1734-1740. Zhu, Z., Xu, Y., Zhao, J., Liu, Q., Feng, W., Fan, J., Wang, P., 2015. miR-367 promotes epithelial-tomesenchymal transition and invasion of pancreatic ductal adenocarcinoma cells by targeting the Smad7-TGF-β signalling pathway. Br. J. Cancer 112, 1367-1375.

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KRAS*: mutational activation of KRAS; RAF: Raf/mil family of serine/threonine protein kinases; MEK: mitogen activated protein kinase; ERK: extracellular signal-regulated kinases; PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase; PDK1: pyruvate dehydrogenase kinase 1; AKT: v-akt murine thymoma viral oncogene homolog; MDM2: mouse double minute 2 homolog; NF-κB: nuclear factor κ B.

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Fig. 2. MiRNAs mediated deregulation of Hedgehog signaling pathway in PDAC.

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HH: hedgehog; PTCH: patched protein; SMO: smoothened; GliA: activated Gli.

Fig. 3. MiRNAs mediated deregulation of canonical TGF-β pathway in PDAC. TGFβR1: TGF-β type 1 receptor; TGFβR2: TGF-β type 2 receptor; TF: transcription factor.

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Fig. 4. MiRNAs mediated signaling network of canonical Wnt pathway in PDAC.

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sFRP2: secreted frizzled related protein 2; Dkk1: Dikkopf-1; FOXM1: proto-oncogene forkhead box M1; APC: adenomatosis polyposis coli; Dvl: dishevelled; GSK-3β: glycogen synthase kinase 3β; TCF: T cell factor; LEF: lymphoid enhancer factor; LRP: low-density lipoprotein receptor related protein. Fig. 5. MiRNAs in the control of cell cycle progression of PDAC.

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CDK: cyclin-dependent kinase; cdc: cell division cycle. Fig. 6. MiRNAs mediated signaling network of apoptosis pathways in PDAC. FADD: Fas (TNFRSF6)-associated via death domain; Bid: BH3 interacting domain death agonist; Bax: Bcl-2-associated X protein; Bcl-2: B-cell CLL/lymphoma 2; Apaf-1: apoptotic peptidase activating factor 1; PDCD4: programmed cell death 4; ING: inhibitor of growth family; NF-κB: nuclear factor κB; NKRF: NF-κB repressing factor; CYLD: cylindromatosis; NFKBIA: nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α. 28

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Hedgehog signaling Wnt signaling

Expression profile

Reference

miR-217

KRAS

Down-regulated

Zhao et al., 2010

miR-206

KRAS and ANXA2

Down-regulated

Keklikoglou et al., 2014

miR-143

Down-regulated

Hu et al., 2012

miR-126

ARHGEF1 (GEF1), ARHGEF2 (GEF2) and KRAS KRAS ADAM9

Down-regulated Down-regulated

Jiao et al., 2012 Hamada et al., 2012

miR-96

KRAS

Down-regulated

Yu et al., 2010

let-7

KRAS and HMGA2

Down-regulated

Watanabe et al., 2009 Torrisani et al., 2009

miR-143/145

KRAS and RREB1

miR-200 family (miR429, -200a, -200b, 200c, and-141) and miR-205 miR-200 family

ZEB1 and SIP1

miR-655 miR-483-3p miR-421

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Kent et al., 2010

Down-regulated

Gregory et al., 2008

Izumchenko et al., 2014

ZEB1 and TGFBR2

Down-regulated

Spaderna et al., 2008

DPC4 and SMAD4 DPC4 and SMAD4

Up-regulated Up-regulated

Hao et al., 2011 Hao et al., 2011

miR-367

SMAD7

Up-regulated

Zhu et al., 2015

miR-212

PTCH1

Up-regulated

Ma et al., 2014

miR-3548 miR-494

Gli-1 FOXM1

Down-regulated Down-regulated

Tsuda et al., 2006 Li et al., 2014

sFRP2, Dkk1 and Kremen2

Up-regulated

Nagano et al., 2013

FRAT2, LRP6, FZD4 and FZD5 FZD5, TMEM92 and HNF1B

Down-regulated Up-regulated

Kapinas et al., 2010 Listing et al., 2015

MIG6

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miR-29c miR-23a/24 miR-34a

CDK6

Down-regulated

Lodygin et al., 2008

miR-107 miR-183

CDK6 Bmi-1

Down-regulated Down-regulated

Lee et al., 2009 Zhou et al., 2014

miR-210

E2F3

Down-regulated

Chen et al., 2012

miR-155 miR-203

BACH-1 and LDOC1 p53

Up-regulated Up-regulated

Greither et al., 2010 Greither et al., 2010

miR-222

p27 (Kip1) and p57 (Kip2)

Up-regulated

Greither et al., 2010

miR-106b miR-148b

p21 and CDKN1A AMPKa1

Up-regulated Down-regulated

Ivanovska et al., 2008 Zhao et al., 2013

miR-148a

CDC25B

Down-regulated

Liffers et al., 2011

miR-132/212 miR-196a

Rb1 NFKBIA

Up-regulated Up-regulated

Park et al., 2011 Huang et al., 2014

miR-26a

cyclin E2

Down-regulated

Deng et al., 2013

miR-219-1-3p miR-181b

MUC4 Bcl-2

Down-regulated Down-regulated

Lahdaoui et al., 2015 Cai et al., 2013

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Down-regulated

Down-regulated

miR-29a

Control of cell cycle

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Target gene

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miRNA

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Signaling pathway KRAS signaling

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Bcl-2

Up-regulated

Dong et al., 2011

miR-15b/16 miR-148a

Bcl-2 CCKBR and Bcl-2

Down-regulated Down-regulated

Shen et al., 2012 Zhang et al., 2014

miR-491-5p

TP53 and Bcl-XL

Down-regulated

Guo et al., 2012

miR-15a-3p miR-17-5p

Bcl-XL Bim

Down-regulated Up-regulated

Druz et al., 2013 Yan et al., 2012

mir-301a

Bim

Up-regulated

Chen et al., 2012

miR-365 miR-375

SHC1and BAX PDK1

Up-regulated Down-regulated

Hamada et al., 2014 Zhou et al., 2014

miR-615-5p

AKT2

Down-regulated

Sun et al., 2015

miR-630 miR-497

IGF-1R IGF-1R

Down-regulated Down-regulated

Farhana et al., 2013 Xu et al., 2014

miR-663

eEF1A

miR-216a miR-130b

JAK2 STAT3

miR-34a

p53

miR-155 miR-371-5p

SC

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miR-21

Zang et al., 2015

Down-regulated Down-regulated

Wang et al., 2014 Zhao et al., 2013

Down-regulated

Chang et al., 2007

TP53INP1 ING 1

Up-regulated Up-regulated

Gironella et al., 2007 He et al., 2014

miR-214

ING 4

Up-regulated

Zhang et al., 2010

miR-196a

ING 5

Up-regulated

Liu et al., 2013

M AN U

Down-regulated

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Table 1. MiRNAs regulating signaling pathways involved in pancreatic tumorigenesis.

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