Carcinogenesis, 2015, Vol. 36, No. 6,

Carcinogenesis, 2015, Vol. 36, No. 6, 676 684 doi:10.1093/carcin/bgv027 Advance Access publication April 11, 2015 Original Manuscript original manuscript mir-29c suppresses pancreatic cancer liver metastasis in an orthotopic implantation model in nude mice and affects survival in pancreatic cancer patients Yongkang Zou, Jianwei Li, Zhiyu Chen, Xiaowu Li, Shuguo Zheng, Dong Yi, Ai Zhong, and Jian Chen * Department of Hepatobiliary Surgery Institute, Southwest Hospital, Third Military Medical University, Chongqing 400038, People s Republic of China * To whom correspondence should be addressed. Tel: +86 23 68765800; Fax: +86 23 65 317637; Email: cjcrj@yahoo.com Abstract We investigated mechanisms of pancreatic cancer metastasis and defined the biological role of mir-29c in pancreatic cancer metastasis. After two rounds of cell selection in vivo, pancreatic cancer cells with various metastatic potentials derived from spontaneous liver metastases were used as a model of pancreatic cancer to determine the role of mir-29c in pancreatic cancer metastasis. Pancreatic cancer samples were analyzed for mirna-29c expression, and these levels were associated with survival between groups. mir-29c suppresses cell migration and invasion by targeting the MMP2 3 UTR. Overexpression of mir-29c suppresses pancreatic cancer liver metastasis in a nude mouse orthotopic implantation model. mir-29c expression was associated with metastasis and pancreatic cancer patient survival. mir-29c plays an important role in mediating pancreatic cancer metastasis to the liver by targeting MMP2. Therefore, mir-29c may serve as a novel marker of pancreatic cancer metastasis and possibly as a therapeutic target to treat pancreatic cancer liver metastasis. Introduction Pancreatic cancer remains as one of the leading causes of cancer deaths and has a 5-year survival rate of <5% (1,2). Pancreatic cancers rapidly metastasize, most commonly to the liver and peritoneum (3). Thus, there is an urgent need to identify sensitive and specific biomarkers for early detection and prognosis of pancreatic cancer. The effects of metalloproteinases, especially MMP2, on the extracellular matrix are very well established, as is the critical role MMP2 plays in cancer metastasis (4 7). MMP2 is overexpressed in a variety of primary malignancies, including pancreatic cancer (8 11). Many studies suggest that MMP2 overexpression is related to increased tumor invasion and metastasis (12,13). As a result, MMP2 expression can be used as a prognostic marker for gastric cancer patients (14). In breast cancer, MMP2 expression correlates with increased metastasis and overall poor prognosis, which suggests this protein is a potent oncogenic factor (15,16). The role of MMP2 in tumor metastasis has attracted attention from many researchers. Our previous study indicated that MMP2 remodels the microenvironment to facilitate metastasis in pancreatic cancer (17). However, the molecular mechanisms underlying its upregulation in pancreatic cancer remain unclear. MicroRNAs (mirna) are endogenous non-coding RNAs between 20 and 25 nucleotides in length. mirnas can negatively regulate gene expression by binding to partially complementary sequences in specific target mrna 3 UTRs (untranslated regions), leading to either mrna degradation or translation inhibition (18,19). The mir-29 family consists of three members: mir-29a, mir-29b and mir-29c (20). A recent study showed that the mir-29 family suppresses invasion and metastasis in hepatocellular carcinoma, lung cancer and prostate cancer by targeting TNFAIP3, MMP2 and Snail (21 23). To investigate the roles of the mir-29 family and MMP2 in pancreatic cancer, we initially analyzed the relationships between the mir-29 family members and MMP2 using in vitro studies and patient samples. We then showed that overexpressed mir-29c can directly bind the MMP2 Received: November 27, 2014; Revised: February 26, 2015; Accepted: March 17, 2015 The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com. 676

J.Chen et al. 677 Abbreviations: mirna TNM UTR MicroRNAs tumor-nodes-metastasis untranslated regions 3 UTR to decrease MMP2 expression and suppress cell migration and invasion. Finally, we examined a spontaneous liver metastasis model of pancreatic cancer to further demonstrate that mir-29c suppressed pancreatic cancer metastasis by downregulating MMP2 expression. Materials and methods Cell lines and clinical tissue samples The human pancreatic cancer cell lines PANC-1, BxPc-3, Hs766t, cfpac-1 and Capan1 were obtained from the American Type Culture Collection. Human pancreatic cancer samples were collected from 107 patients after surgical resection at Southwest Hospital, The Third Military University, ChongQing, China, between January 2010 and December 2013. Among the 107 patients, 2 patients died during the perioperative period because of surgery-related complications. To avoid any surgery-related confounder effects, these two patients were excluded from the survival analysis. Ultimately, 105 cases were included in the univariate and multivariate Cox regression analyses in our manuscript. Informed consent was obtained from the patients before sample collection. The samples were immediately snap-frozen in liquid nitrogen or fixed in 10% formalin for paraffin embedding. Clinical pathological data were collected from the patients medical records. Patient survival data were collected using telephone interviews every 3 months after resection until death or the end of data collection for this study (December 2013). Patient characteristics are listed in Table 1. mirna extraction and quantification mirna and mrna extraction from pancreatic cancer cells was performed using the TRIZOL reagent. The formalin-fixed, paraffin-embedded slides were deparaffinized, rehydrated and then stained with hematoxylin and eosin to identify the pancreatic cancer and adjacent tissues. The pancreatic cancer and adjacent tissues were then scraped with a fine scalpel and transferred into Eppendorf tubes. RNA was extracted using the mirneasy FFPE Kit (Qiagen). mir-29c expression was determined by qrt-pcr using SYBR Green Dye (Applied Biosystems). Expression levels were normalized against an endogenous U6 mrna control. The relative expression of mir- 29c in each paired pancreatic cancer tissue and adjacent tissue was calculated using the 2 ΔΔCT method. Cell transfection The PANC-1, BxPc-3 and Capan1 cells were cultured to 80% confluence in sixwell plates. The cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer s instructions. The cells in each well were transfected with 100 nm mimics or 200 nm inhibitor (Ribobio, GuangZhou, China). After 24 and 48 h of transfection, the expression levels of mir-29c and MMP2 were determined using qrt-pcr and western blot analyses. The cells were also used in proliferation, migration and invasion assays. Western blot analysis Cell lysates were prepared by extracting proteins with radio-immunoprecipitation assay buffer containing a protease inhibitor cocktail (Upstate, Lake Placid, NY) according to the manufacturer s instructions. The anti- MMP-2 monoclonal antibody and an antiactin antibody (Santa Cruz) were used at 1:1000 and 1:5000 dilutions, respectively. Cell proliferation assays Cell proliferation was measured using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). According to the manufacturer s instructions, 2 10 3 cells were seeded in a 96-well plate containing 100 µl of medium in each well. Cells were cultured for 24, 48, 72 and 96 h, and the CCK-8 solution (10 μl) was added to each well, followed by incubation at 37 C for 2 h. The absorbance was measured at 460 nm to calculate the number of cells present. Migration and invasion assay Migration and invasion assays were performed in a 24-well chamber containing a polycarbonate membrane with 8-μm pores (Corning, NY). For the migration assays, cells were only applied to the transwell chamber without Matrigel (R&D). For the invasion assays, the membrane was coated with 10 μg of Matrigel to form a matrix barrier. Tumor cells (3 10 4 in 100 μl of serum-free medium) were added to the upper compartment of the chamber, and the lower compartment was filled with 500 μl of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. After incubation at 37 C for 12 or 24 h, the cells remaining on the upper membrane surface were removed. The cells on the lower membrane surface were fixed, stained with crystal violet and then counted using a light microscope. Gelatin zymography Gelatin zymography for MMP2 was performed as previously described (17). The conditioned medium was collected from serum-free culture conditions and subjected to gelatin- sodium dodecyl sulfate polyacrylamide gel electrophoresis electrophoresis. The gel contained 1% gelatin and 30% acrylamide. The gels were treated with 2.5% Triton X-100 at 37 C for 30 min to remove the sodium dodecyl sulfate and were then incubated at 37 C for 16 h in substrate buffer (5 mmol/l CaCl 2, 50 mmol/l Tris HCl, ph 8.0). The gels were stained with 0.15% Coomassie blue R250 (Bio-Rad) in 50% methanol/10% glacial acetic acid at room temperature for 30 min. The gels were then destained in the same solution without Coomassie blue. MMP2 activity was identified as clear gelatin-degraded bands against the blue background. Ductal luciferase reporter assays PANC-1 cells (5 10 4 ) were seeded in a 48-well plate. The cells were cotransfected with 10 nm mir-29c or NC mimic and 10 ng of a firefly luciferase reporter construct. The reporter contained either a wild-type or mutant MMP2 3 UTR. The transfections were performed in duplicate, and threeindependent experiments were performed. The luciferase activities were analyzed 48 h after transfection using the Dual-Luciferase Reporter Assay System (Promega) in an M200 microplate fluorescence reader (Tecan, Vienna, Austria). Orthotopic model of pancreatic cancer For in vivo tumor experiments, 6- to 8-week-old male nude mice weighing 20 25 g were obtained from DaPing Hospital (Chongqing, China). All experiments were conducted in accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals. All surgical instruments were autoclaved and allowed to cool to room temperature before use. The mice were anesthetized intraperitoneally with 1% pentobarbital sodium (50 mg/kg). Their abdomens were disinfected with alcohol, and an incision was created in the left upper abdomen and peritoneum. The pancreas was carefully exposed, and the tail of the pancreas was lifted using a cotton swab. The tail of the pancreas was then injected with 5 10 5 tumor cells. The pancreas was then replaced in the abdominal cavity, and the abdomen was closed with two layers with 5 0 absorbable sutures. Metastasis was analyzed using live imaging and magnetic resonance imaging. Hs766t-L2 cells were initially transfected with a mir-29c agomir (200 nm) for 48 h or an agomir negative control (200 nm). For the orthotopic model of pancreatic cancer, 5 10 5 cells were injected into two groups (10 mice/group) of 6- to 8-week-old nude mice. The two groups of mice were treated with mir-29c agomir (5 nmol each) or a negative control agomir (5 nmol each) by intraperitoneal injection twice a week for 2 weeks. The treatments began on week 3 after tumor inoculation. Immunohistochemical analysis Formalin-fixed, paraffin-embedded tissues were cut in 5-μm sections. Following deparaffinization, the sections were rehydrated and subjected to antigen retrieval by microwaving in 0.01 M sodium citrate (ph 6) for 10 min. The sections were incubated at 4 C overnight with monoclonal antibodies raised against MMP2 (Proteintech). The protein staining was evaluated using light microscopy at 400 magnification. The staining was subjectively graded as negative (<25%), weak (25 50%), moderate (50 75%) or intense (>75%), depending on the percentage of positively stained tumor cells.

678 Carcinogenesis, 2015, Vol. 36, No. 6 Table 1. Association of mir-29c and MMP2 expression with clinicopathological characteristics of pancreatic cancer patients mir-29c expression MMP2 expression Low expression, N = 50 High expression, N = 57 P value Low expression, N = 45 High expression, N = 62 P value Age a 0.111 0.877 <56 years 27 22 21 28 56 years 23 35 24 34 Gender 0.086 0.987 Male 32 45 32 45 Female 18 12 13 17 Tumor differentiation 0.002 0.007 1 2 31 50 40 41 3 19 7 5 21 TNM stage I II 20 56 45 31 III/IV 30 1 0 31 Nodal metastasis 0 15 46 44 17 1 35 11 1 45 Tumor size 0.658 0.758 <2 cm 16 16 14 18 >2 cm 34 41 31 44 TNM, tumor-nodes-metastasis. All analyses were conducted using χ 2 tests. Low versus high mir-29c expression was determined according to the cut-off values for mir-29c, which were defined as the cohort median. Low versus high MMP2 expression was determined according to staining grade: negative and weak staining represented low expression, whereas moderate and intense staining represented high expression. a For age, the median value for all patients was used as the cut-off point to define the subgroups (<56 years old and 56 years old groups). Statistical analysis All data were analyzed using SPSS 13.0. The association between mir- 29c and MMP2 protein expression in pancreatic cancer cells was analyzed using Spearman s correlation. The correlations between mir-29c/ MMP2 and pathological parameters were determined using χ 2 tests. The Kaplan Meier method was used to compare overall survival among the different patient groups. The log-rank test was used to estimate differences in survival. Univariate and multivariate analyses were based on the Cox proportional hazards regression model. A P < 0.05 was considered to be statistically significant. Results mir-29c and MMP2 correlate with pancreatic cancer cell differentiation To determine whether mirnas participate in the regulation of MMP2 expression, we used TargetScan (http://www.targetscan. org/) and PicTar (http://pictar.mdc-berlin.de/) software to predict MMP2-targeting mirnas. Only the mir-29 family members were predicted by both algorithms. To investigate the potential relationship between the mir-29 family members and MMP2, we first analyzed their expression in pancreatic cancer-derived cell lines representing different tumor grades. Therefore, we initially analyzed mir-29 expression in five pancreatic cancer cell lines with different tumor differentiation grades by RT PCR (24,25). As shown in Figure 1A, only mir-29c, but not mir-29a and mir-29b, was inversely correlated with the pancreatic cancer cell tumor grade (Supplementary Table 1, available at Carcinogenesis Online). We then evaluated MMP2 expression and activity in five pancreatic cancer cell lines using RT PCR, western blot and zymography analyses (Figure 1B). Our data showed that the highly differentiated pancreatic cancer cell lines Capan1 (G1), Hs766t (G2) and cfpac (G2) expressed relatively low levels of MMP2. However, the less differentiated pancreatic cancer cell lines BxPc-3 and PANC1 (G3) showed relatively high levels of MMP2 expression. The less differentiated PANC1 (G3) cells also exhibited high MMP2 activity, whereas the highly differentiated CAPAN1 (G1), Hs766t (G2) and cfpac (G2) cells had very low MMP2 activity. Thus, mir-29c expression was inversely correlated with MMP2 expression in pancreatic cancer cells (Figure 1C). These results suggest that mir-29c and MMP2 expression correlate with pancreatic cancer cell differentiation. mir-29c suppresses cell migration and invasion by regulating MMP2 expression To determine whether mir-29c upregulation reduces MMP2 expression and suppresses the migration and invasion of pancreatic cancer cells, we transfected PANC-1 and BxPc-3 cells with mir-29c mimics or a negative control and then evaluated cell proliferation, MMP2 expression, migration and invasion. The real-time PCR data in Figure 2A confirm successful mir-29c overexpression. PANC-1 and BxPc-3 cells overexpressing mir- 29c had significantly decreased levels of MMP2 expression and activity compared with the negative control (Figure 2B). This overexpression also significantly decreased cell migration and invasion (Figure 2C). The Capan1 cell line expresses relatively high levels of endogenous mir-29c. Therefore, we asked whether mir-29c downregulation induces MMP2 expression. MMP2 expression, migration and invasion were significantly increased when Capan1 cells were transiently transfected with mir-29c inhibitors. Furthermore, Figure 2D shows that sirna-mediated knockdown of MMP2 in PANC-1 cells suppressed migration and invasion. However, neither mir-29c overexpression nor mir-29c inhibitors affected the migration or invasion of PANC si-mmp2 cells (Figure 2E). These results indicate that mir-29c suppresses cell migration and invasion by regulating MMP2 expression. mir-29c directly binds to the MMP2 3 UTR to regulate MMP2 expression The 3 UTR of MMP2 mrna contains one binding site for mir- 29c. To examine whether mir-29c regulates MMP2 expression

J.Chen et al. 679 by directly binding to the MMP2 3 UTR, we cotransfected a mir29c mimic with a dual-luciferase reporter construct containing either the wild-type or a mutant MMP2 3 UTR. We found that in the wild-type MMP2 3 UTR-transfected cells, the mir-29c mimic significantly decreased luciferase activity. In contrast, in mutant MMP2 3 UTR-transfected cells, the mir-29c mimic did not reduce luciferase activity (Figure 3). Therefore, our data suggest that mir-29c can directly bind to the MMP2 3 UTR and regulate MMP2 expression. mir-29c suppresses pancreatic cancer metastasis by downregulating MMP2 expression in vivo To obtain pancreatic cancer cells with different metastatic potentials in vivo, we used the cell lines that express high levels of mir-29c, Capan1 and Hs766t. The cells were labeled with luciferase and injected into the pancreases of nude mice. We then isolated, expanded and reinjected the spontaneous liver metastases into the pancreases of secondary recipients. Only Hs766t cells successfully developed spontaneous liver metastases after 12 weeks. The second generation of Hs766t cells (Hs766t-L2) developed liver metastases in 8/10 mice, and the first generation of Hs766t cells (Hs766t-L1) developed liver metastases in 5/10 mice. In contrast, parental Hs766t cells developed liver metastases in 2/10 mice (Figure 4A right, Supplementary Table 2, available at Carcinogenesis Online). Therefore, we successfully isolated pancreatic cancer cells with different metastatic potentials. We then examined whether mir-29c and MMP2 were involved in the differential metastatic potential of these three pancreatic cancer cell lines. Our data indicate that mir-29c expression was significantly decreased and MMP2 expression was significantly elevated in Hs766t-L2 and Hs766t-L1 cells compared with Hs766t cells (Figure 4B). These results indicated that mir-29c and MMP2 were involved in the metastatic potential of pancreatic cancer cells. To further address whether mir-29c functions through MMP2 and affects pancreatic cancer metastasis, we overexpressed mir-29c in Hs766t-L2 cells and determined its effects on MMP2 expression and in vivo pancreatic cancer liver metastasis. We found that mir-29c overexpression significantly suppressed MMP2 expression. Ex vivo luciferase imaging and magnetic resonance imaging both revealed that mir-29c overexpression significantly reduced liver metastasis in Hs766t-L2 cells compared with the negative control (Figure 4C). In orthotopic xenografts, mir-29c expression increased, and MMP2 expression significantly decreased in the mir-29c overexpressing group compared with the negative control. However, mir-29c expression remained high, and MMP2 expression was not reduced in the mir-29c overexpression group (Figure 4D); thus, another mechanism may regulate MMP2 expression. Therefore, mir-29c downregulates MMP2 expression and can suppress pancreatic cancer metastasis. Figure 1. mir-29c expression correlates with higher differentiation and lower MMP2 expression. (A) The expression of the mir-29 family members as determined by RT PCR in five pancreatic cancer cell lines with different tumor grades. (B) MMP2 expression and activity in five pancreatic cancer cell lines as determined by RT PCR, western blotting and zymography analyses. (C) mir-29c expression was inversely correlated with MMP2 protein expression in pancreatic cancer cells.

680 Carcinogenesis, 2015, Vol. 36, No. 6 Figure 2. mir-29c suppresses cell migration and invasion by regulating MMP2 expression. (A) Re-expression of mir-29c was confirmed by q-pcr (Left), MMP2 mrna and protein expression and zymography. (B) Re-expression of mir-29c significantly reduced cell migration and invasion in PANC-1 and BxPc-3 cells. (C) A mir-29c inhibitor enhanced Capan1 cell migration and invasion. (D) MMP2 mrna and protein were detected after transfection with si-mmp2-1, si-mmp2-2 or si-mmp2-3 (Left). MMP2 knockdown with si-mmp2-3 significantly suppressed PANC-1 pancreatic cancer cell migration. Re-expression of mir-29c profoundly reduced cell migration in PANC-1 si-mmp2-3 control cells but had no effect on PANC-1 si-mmp2-3 cells (Right). *P < 0.05 was considered statistically significant.

J.Chen et al. 681 Figure 3. Relative luciferase activity. mir-29c significantly suppressed the luciferase activity of the MMP2 wild-type 3 UTR construct but not the mutant 3 UTR construct. The Renilla luciferase vector was used as an internal control. *P < 0.05 was considered statistically significant. mir-29c and MMP2 expression levels associate with pancreatic cancer patient prognosis We further analyzed the relationship between mir-29c and MMP2 in 107 pancreatic cancer tissues and corresponding paracancerous tissues using qrt-pcr and immunohistochemical analyses. Our results showed that mir-29c expression was significantly lower in pancreatic cancer tissues compared with pair-matched adjacent paracancerous tissues (Figure 5A). In contrast, MMP2 was frequently overexpressed in pancreatic cancer tissue compared with paracancerous tissues. mir-29c expression was also inversely associated with MMP2 expression in pancreatic cancer tissues (Figure 5B). The clinicopathological analyses of 107 patient tissues showed that decreased mir-29c expression was significantly correlated with lymph node metastasis, tumor differentiation and TNM stage. There was no significant correlation between mir-29c expression and age, gender or tumor size. In contrast, increased MMP2 expression significantly correlated with lymph node metastasis, tumor differentiation and TNM stage (Table 1). However, of the 46 cases with nodal metastasis, 11 cases exhibited high levels of mir-29c and MMP2 expression. To determine the prognostic significance of mir-29c, we analyzed the correlation between mir-29c expression and pancreatic cancer patient survival. We found that low mir-29c expression or high MMP2 expression was significantly associated with a poor overall survival rate (Figure 5C and D). The univariate analysis showed that mir-29c, MMP2, tumor size and TNM stage were significantly associated with overall survival in pancreatic cancer patients. A multivariate analysis showed that mir-29c, MMP2, TNM and node metastasis were independent prognostic indicators for overall survival (Table 2). Finally, a combined mir-29c and MMP2 expression analysis showed that patients with low levels of mir-29c and high levels of MMP2 had the lowest overall survival. In contrast, pancreatic cancer patients with high mir-29c expression and low MMP2 expression had the best overall survival (Figure 5E, Supplementary Table 3, available at Carcinogenesis Online). These results suggest that mir-29c downregulation is associated with increased MMP2 expression and pancreatic cancer metastasis. Therefore, the expression levels of these genes may predict pancreatic cancer patient survival. Discussion Our data indicate that mir-29c suppresses human pancreatic cancer cell invasion and metastasis in an orthotopic implantation model in nude mice and that mir-29c affects metastasis and survival in pancreatic cancer patients via the negative regulation of MMP2. These conclusions were drawn from multiple observations: (i) We successfully isolated two pancreatic cancer cell lines, Hs766t-L1 and Hs766t-L2, with enriched liver metastasis by orthotopic selection in vivo. (ii) mir-29c expression was significantly decreased in Hs766t-L2 and Hs766t-L1 cells compared with the parental Hs766t cells. (iii) mir-29c overexpression significantly reduced pancreatic cancer liver metastasis in an orthotopic implantation model in nude mice compared with the negative control. (iv) mir-29c directly binds to the MMP2 3 UTR and regulates MMP2 expression in pancreatic cancer cells. (v) mir-29c downregulation is associated with overall survival and metastasis in pancreatic cancer patients. The suppressive effects of mir-29s expression on invasion and metastasis have been demonstrated in gastric cancer, hepatocellular carcinoma and lung cancer (22,26 28). However, the roles of the mir-29 family members have not been analyzed in pancreatic cancer. In this study, we found that mir-29c, but

682 Carcinogenesis, 2015, Vol. 36, No. 6 not mir-29a or mir-29b, suppressed invasion and metastasis by downregulating MMP2 expression. However, in oral squamous cell carcinoma, mir-29a upregulates MMP2 to promote cancer invasion (29), whereas mirna-29b suppresses invasion and metastasis by targeting MMP2 in hepatocellular carcinoma (27). Previous studies identified pancreatic cancer metastasis-related mirnas based on data obtained from the differential mirna expression profiles of tumors compared with paracancerous tissues. However, some potential metastasisrelated genes may be neglected. In our study, we successfully isolated two pancreatic cancer cell lines that exhibited enriched metastatic activity to the liver through in vivo selection in an orthotopic implantation model. We further demonstrated that mir-29c expression is significantly correlated with the potential for pancreatic cancers to metastasize to the liver. This is an intriguing observation and is a topic of further investigation to Figure 4. mir-29c suppresses pancreatic cancer metastasis by downregulating MMP2 expression in vivo. (A) Schematic representation of the in vivo selection process. Hs766t cells were inoculated into the pancreases of nude mice. Liver metastases were detected by live imaging analysis after 10 12 weeks. The tumor cells were isolated from liver metastases and re-inoculated after expansion in culture. After two rounds of selection from the metastases, pancreatic cancer cells with different metastatic potentials, Hs766t-L1 and Hs766t-L2, were isolated. The liver metastasis potential is shown in the top right panel. (B) mir-29c and MMP2 expression in pancreatic cancer cells with three different metastatic potentials: Hs766t, Hs766t-L1 and Hs766t-L2. (C) Twelve weeks postorthotopic implantation in nude mice, liver metastases derived from mir-29c agomir- and control-transfected Hs766t-L2 cells were observed by live imaging and magnetic resonance imaging. A representative liver metastasis is shown in the bottom right panel. (D) mir-29c and MMP2 expression in orthotopic xenografts and liver metastases were determined by RT PCR and immunohistochemistry. *P < 0.05 and **P < 0.01 were considered statistically significant.

J.Chen et al. 683 Table 2. Univariate and multivariate analyses of factors associated with the overall survival of pancreatic cancer patients Variables Univariate analysis Age (<56 years versus 56 years) Gender (male versus female) mir-29c (high versus low) MMP2 (high versus low) Tumor differentiation Tumor size TNM stage Nodal metastasis Multivariate analysis mir-29c (high versus low) MMP2 (high versus low) TNM stage Nodal metastasis Case number Hazard ratio (95% CI) P value 57/48 76/29 55/50 60/45 79/26 74/31 74/31 60/45 1.057 (0.686 1.630) 1.062 (0.660 1.771) 0.687 (0.615 0.716) 2.657 (2.072 3.408) 2.601 (1.560 4.334) 0.848 (0.529 1.359) 17.051 (8.522 34.075) 9.973 (5.828 17.065) 0.790 0.793 0.467 55/50 60/45 74/31 60/45 0.880 (0.775 1.000) 1.688 (1.164 2.447) 3.035 (1.241 7.423) 2.373 (0.980 5.749) identify more metastasis-related gene in Hs7667, Hs766t-L1 and Hs766t-L2 cells. Current animal models of pancreatic cancer include carcinogen-induced models (30), genetically engineered models (31) and xenograft models (32). When pancreatic cancer is induced in carcinogen induced models, hepatocellular carcinoma and other tumors occur simultaneously due to poor specificity of the carcinogen; thus, the accuracy of using such animal models is limited. A genetically engineered model was recently developed that is particularly useful for the study of certain genes in tumorigenesis and tumor progression. However, such models are not suitable for tumor metastasis in animals due to their technical requirements and huge expense. Xenograft models are particularly applicable for research on pancreatic cancer metastasis. Two main types of xenograft model are available: subcutaneous transplantation and orthotopic transplantation. The orthotopic transplantation model is more relevant given that it simulates local invasion, survival in circulation and distant metastasis in a manner similar to human pancreatic cancer metastasis. Establishing orthotopic transplantation models and isolating pancreatic cancer cells with varying metastatic potentials is an effective model for metastasis research in pancreatic cancer. Clinically, our data reveal that low levels of mir-29c expression were associated with high MMP2expression, lymph node metastasis, tumor differentiation and TNM stage. More importantly, Figure 5. Lower mir-29c expression and higher MMP2 expression are associated with poor prognosis. (A) A comparison of mir-29c expression between pancreatic cancer and the paracancerous tissues. A log2-fold change less than 0 was considered as downregulated. (B) MMP2 protein expression was examined by immunohistochemistry, and the correlation between MMP2 expression and mir-29c expression was assessed in 107 pancreatic duct adenocarcinoma patients. Representative immunohistochemistry images are shown. The original magnification is 400. The statistical significance between the difference in MMP2 expression and mir-29c low or mir-29c high expression was calculated with using Student s t-test. (C) Patients with higher mir-29c expression had better overall survival, (D) and patients with higher MMP2 expression had poorer overall survival in pancreatic cancer tissues. (E) The overall survival was calculated for the four groups, which were divided according to mir-29c and MMP2 expression (group I, low mir-29c/high MMP2; group II, low mir-29c/low MMP2; group III high mir-29c/low MMP2 and group IV high mir-29c/high MMP2).

684 Carcinogenesis, 2015, Vol. 36, No. 6 our clinical data also suggested that mir-29c and MMP2 were independent prognostic indicators for overall survival in pancreatic cancer patients. Patients with low mir-29c and high MMP2 expression had the worst overall survival. These results suggest that mir-29c and MMP2 are independent prognostic markers for pancreatic cancer patients but that the combination of mir-29c and MMP2 has a stronger prognostic value. We also report that mir-29c is highly expressed in non-metastatic pancreatic cancer tissues. These data indicate that it might be possible to use mir- 29c to predict the likelihood of pancreatic cancer metastasis. Metastasis is a complex series of steps in which cancer cells leave the original tumor site and migrate to a distant organ. Certain cancers tend to spread to specific organ sites; however, the underlying mechanism of metastasis is not completely understood. Different gene signatures may determine different organ targets through the involvement of the metastatic microenvironment. For example, different sets of genes were identified as mediating breast cancer metastasis to lung, bone and brain (33 35). A similar phenomenon was also identified regarding colon cancer metastasis to liver and lung (36). Whether the same phenomenon exists for pancreatic cancer liver metastasis requires further research. In our study, we successfully isolated two specific pancreatic cancer cell lines with enriched metastatic activity to the liver through orthotopic implantation and in vivo selection. These cells will help to identify a set of genes that mediate pancreatic cancer metastasis to the liver. Therefore, these data suggest that mir-29c may play an important role in metastasis and may be a novel prognostic marker and potential therapeutic target in pancreatic cancer. This study opens new avenues for understanding the mechanism underlying pancreatic cancer liver metastasis and for the development of effective therapeutics for pancreatic cancers. Supplementary material Supplementary Tables 1 3 can be found at http://carcin.oxfordjournals.org/ Funding National Natural Science Foundation of China (81071975); Project of State Key Laboratory of Hepatobiliary Surgery at Southwest Hospital. Conflict of Interest Statement: None declared. References 1. Siegel, R. et al. (2012) Cancer statistics, 2012. CA. Cancer J. Clin., 62, 10 29. 2. Li, D. et al. (2004) Pancreatic cancer. Lancet, 363, 1049 1057. 3. Cascinu, S. et al. (2010) Pancreatic cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann. Oncol., 21 (suppl. 5), v55 v58. 4. Kessenbrock, K. et al. (2010) Matrix metalloproteinases: regulators of the tumor microenvironment. Cell, 141, 52 67. 5. Jezierska, A. et al. 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