Tumor Biology

, Volume 33, Issue 6, pp 1863–1870

miR-143 inhibits the metastasis of pancreatic cancer and an associated signaling pathway

Authors

  • Yongjun Hu
    • Department of General Surgery, Xiangya HospitalCentral South University
  • Yanglu Ou
    • Department of General Surgery, Xiangya HospitalCentral South University
  • Kemin Wu
    • Department of General Surgery, Xiangya HospitalCentral South University
  • Yuxiang Chen
    • Hepatobiliary and Enteric Surgery Research Center, Xiangya HospitalCentral South University
    • Department of General Surgery, Xiangya HospitalCentral South University
Research Article

DOI: 10.1007/s13277-012-0446-8

Cite this article as:
Hu, Y., Ou, Y., Wu, K. et al. Tumor Biol. (2012) 33: 1863. doi:10.1007/s13277-012-0446-8

Abstract

Pancreatic cancer is characterized by early metastasis and high mortality. In this study, the role of miR-143 in invasion and metastasis was investigated in pancreatic cancer cells. miR-143 expression was established by an adenovirus-carried miR-143 expression cassette. mRNA and protein levels of gene expression were examined by RT-PCR and Western blot assay, respectively. Rho GTPases activity was measured by the pull down assay. The role of miR-143 in migration and invasion of Panc-1 cells was tested in vitro. The antimetastatic effect of miR-143 was tested in a liver metastasis model, while its antitumor growth effect was tested in a xenograft Panc-1 tumor model. Results demonstrated that ARHGEF1 (GEF1), ARHGEF2 (GEF2), and K-RAS genes are the targets of miR-143. miR-143 expression significantly decreased mRNA and protein levels of GEF1, GEF2, and K-RAS genes; lowered the constitutive activities of RhoA, Rac1, and Cdc42 GTPases; decreased the protein levels of MMP-2 and MMP-9; but significantly increased the protein level of E-cadherin. miR-143 expression also significantly inhibited the migration and invasion of Panc-1 cells in vitro, liver metastasis, and xenograft tumor growth in vivo. Our study suggested that miR-143 plays a central role in the invasion and metastasis of pancreatic cancer and miR-143 is a potential target for pancreatic cancer therapy.

Keywords

microRNAmiR-143Rho GTPaseGEFE-cadherinPancreatic cancer

Introduction

Pancreatic cancer is the eighth leading cause of death among cancers worldwide [1, 2]. Both the prevalence and incidence of pancreatic cancer are increasing worldwide. Despite recent progress in targeting therapy, overall survival rate of pancreatic cancer patients remains less than 5 % [3, 4]. A major reason for the high mortality of pancreatic cancer is its highly aggressive nature. Invasion and metastasis occur very early in pancreatic cancer [1]. Therefore, metastasis is a sign of poor prognosis in pancreatic cancer. However, key molecules involved in metastasis of pancreatic cancer have not yet been identified.

Metastasis is an extraordinarily complex multistep process that includes detachment of individual tumor cell from the primary tumor, cell motility and migration, establishment of metastatic foci at the secondary site, and arrest of tumor cells by local endothelial cells through adhesive interactions [5]. This process strongly depends on adhesive and invasive properties of tumor cells [6]. Rho GTPases control several molecular events such as reorganization of the actin cytoskeletion, epithelial-cadherin (E-cadherin)-mediated cell–cell contact, and the expression of matrix metalloproteinases (MMPs) [7, 8]. Activation of Rho GTPases leads to an aggressive, highly metastatic cellular phenotype. Overexpression of Rho GTPases, such as RhoA, Rac1, and Cdc42, has been observed in several tumors including pancreatic tumors and was thought to play a crucial role in tumor cell migration and invasion [9]. Rho GTPases are activated by guanine nucleotide exchange factors (GEFs), which promote Rho activity by catalyzing the exchange of GDP for GTP [9]. A recent study demonstrated that the RAS oncogene also regulates Rho GTPase pathways to mediate migration and invasion of human colon cancer cells [10]. However, how GEFs and RAS gene expression are regulated in pancreatic cancer cells have not been elucidated.

Recently, microRNAs (miRNAs) have been identified as both tumor suppressor genes and oncogenes. miRNAs are often abnormally expressed in many types of cancers [11]. However, the functional role of most miRNAs remains unexplored. miR-143 has been demonstrated to function as a tumor suppressor, and loss of miR-143 expression has been reported in many cancer types, whereas restoration of miR143 expression has been shown to abrogate tumorigenesis [12]. The loss of miR-143 expression is also observed frequently in KRAS mutant pancreatic cancer tissues [13] and pancreatic cancer cell lines [14]. miR-143 has been demonstrated to repress the expression of KRAS2 and its downstream effectors [15]. However, only a few miR-143 targets are experimentally verified. Particularly, the role of miR-143 in the metastasis of pancreatic cancer has not been reported.

In this study, we measured the expression of K-RAS, GEF1, and GEF40 genes in miR-143-expressing Panc-1 pancreatic cancer cells. GEF1 and GEF40 genes are two possible targets of miR-143 predicted by TargetScan software. Downstream Rho GTPases' activity, the expression of E-cadherin and the expression of metalloproteinases were also examined after restoration of miR-143 expression.

Materials and methods

Cell culture

Panc-1, a human pancreatic cancer cell line isolated from liver metastases of pancreatic cancer, was obtained from the American Type Culture Collection (ATCC) and grown in RPMI 1640 medium supplemented with l-glutamine, antibiotics, and 10 % fatal bovine serum (Gibco Laboratories, USA). Cells were cultured at 37 °C in 5 % CO2.

Preparation of adenovirus to express miR-143

The miR-143 expression cassette was constructed by placing the hsa-miR-143 minigene sequence, containing complimentary hsa-miR-143 cDNA sequences (5′-TGAGATGAAGCACTGTAGCTC-3′) and a polythymidine tract (TTTTTT), into the pSilencer-2.0 vector (Ambion Inc.). The minigene was engineered to possess BamHI- and HindIII-compatible overhangs to facilitate its ligation. The assembled plasmid, called pSilencer-miR-143, can transcribe a 21-bp miR-143. The control plasmid was pSilencer-NT (NT stands for nontargeted, obtained from Ambion), which is a plasmid with a nontargeted sequence (5′-AATTCTCCGAACGTGTCACGT-3′) that replaces the miR-143 sequence.

The AdEasy system of adenovirus packaging was purchased from Stratagene Corp. (La Jolla, CA, USA). The miR-143 expression cassette, including the U6 gene promoter, miR-143 minigene sequence, and the polythymidine stop sequence was excised by PvuII/HindIII from pSilencer-miR-143 vector and subcloned into the EcoRV/HindIII sites of pAdTrack vector. The resulting plasmid was pAdTrack-U6-miR-143. The adenovirus that carries the miR-143 gene was produced as previously described [16]. The resultant adenoviral vector was named Ad-miR-143. A control adenovirus called Ad-NT was produced with the nontargeted sequence as described above.

Northern blot analysis

Total RNA was extracted using the Trizol reagent (Invitrogen, Grand Island, NY, USA). The miRNA was further isolated using PureLink™ miRNA isolation kit (Invitrogen). One microgram of denatured small RNA was run on a 12.5 % urea–polyacrylamide gel. RNA was then transferred to a nylon membrane. Prehybridization and hybridization were performed with the ExpressHyb buffer (Invitrogen) at 68 °C. The membrane was rinsed and developed for signal detection. The antisense oligonucleotide of miR-143 was 32P-labeled using [γ-32P]ATP and T4 polynucleotide kinase. The 32P-end labeled oligonucleotide probe was used directly for hybridization. The band size of miRNA was determined by the ZR small-RNA™ ladder (Zymo Research, Orange, CA, USA).

RT-PCR

Total RNA was extracted as described above. Reverse transcription was performed using Superscript III reverse transcriptase (Invitrogen) by following the user manual. PCR amplification was carried out using Taq DNA polymerase (Invitrogen) and two pairs of primers in the same reaction: one amplifies the target gene fragment and another amplifies the GAPDH gene serving as an internal control. The PCR cycle number was adjusted to anywhere from 25 to 32 when two gene fragments can be visualized on a 1.5 % agarose gel containing 0.5 μg/ml ethidium bromide. The GEF1 gene was amplified using forward primer: 5′-CCACCTGGAGCAGCCCTG-3′ and reverse primer: 5′-TCCAGCTGCCGGCCCACG-3, producing a 502-bp fragment. The GEF40 gene was amplified using forward primer: 5-CGCCGCCCTGTATCCACC-3′ and reverse primer: 5′-GGCGCAGACAGTAGGCAG-3′, producing a 470-bp fragment. The K-RAS2 gene was amplified using forward primer: 5′-GTAGTTGGAGCTGGTGGC-3′ and reverse primer: 5′-TTTCACACAGCCAGGAG-3′, producing a 520-bp fragment. The GAPDH gene was amplified by the forward primer: 5′-GGTGAAGGTCGGTGTGAACG-3′ and reverse primer: 5′-TGGAGGCCATGTAGGCCATG-3′, producing a 990-bp fragment.

Western blot analysis

Panc-1 cells were infected with Ad-NT or Ad-miR-143 and were then harvested in RIPA buffer containing 1 % protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations were measured using BCA Protein Assay kit (Beyotime, Shanghai, China). Twenty micrograms of total protein was loaded onto a 12 % SDS-PAGE gel and transferred to nitrocellulose membranes. After blocking with 5 % nonfat milk for 1 h, membranes were incubated with primary antibody overnight at 4 °C and subsequently incubated with HRP-labeled secondary antibody (1:2,000 dilution) for 2 h at room temperature the next day. Reactive proteins were detected using chemiluminescent reagents (Pierce, Rockford, IL, USA). To control for loading efficiency, the blots were stripped and reprobed with α-tubulin antibody (1:2,000 dilution, Sigma-Aldrich, St. Louis, MO, USA). The antibodies for E-cadherin (1:1,000 dilution), MMP2 (1:1,000 dilution), MMP9 (1:1,000 dilution), and the peroxidase-labeled secondary antibody were purchased from Cell Signaling Technology (Beverley, MA, USA). The anti-K-RAS (1:1,000 dilution), anti-GEF1 (1:1,000 dilution), and anti-GEF40 (1:1,000 dilution) antibodies were purchased from Abcam (Cambridge, MA, USA).

GTPase activity assays

The activity of RhoA, Rac1, and Cdc42 was examined using the Active Rho, Rac1, and Cdc42 Pull-Down and Detection kits (Pierce, Rockford, IL, USA). Panc-1 cells were seeded in 10-cm dishes (1 × 106 cells) and grown for 12 h. Cells were then infected with 10 MOI of Ad-NT or Ad-miR-143 for 24 h. After cells were homogenized on ice for 10–15 s by sonication, large cell debris was removed by centrifugation at 12,000 g for 5 min at 4 °C. Activated RhoA, Rac1, and Cdc42 were purified using the kits mentioned above. The amount of GTP-bound Rac1, Cdc42, and RhoA as well as the total amount of Rac1, Cdc42, and RhoA in cell lysates were determined by Western blot with their respective antibodies.

Cell migration assay

The migration experiment was performed using a modified Boyden chamber apparatus as previously described [17]. Briefly, Panc-1 cells were infected with Ad-NT or Ad-miR-143 for 24 h. Cells were then harvested and resuspended with RPMI 1640 containing 0.1 % BSA. The cells (1 × 105/100 μl) were loaded into the upper chamber, and 10 ng/ml of epithelial growth factor (EGF) was placed in the lower chamber. A membrane filter separating the chambers with 8 μm pores was coated with 100 μg/ml of rat tail collagen overnight at 4 °C. The number of cells that migrated to the lower surface in 4 h was determined by counting the cells in four places under the microscope at ×400 magnification.

Matrigel invasion assay

The invasion activity of Panc-1 cells was assessed using a Matrigel invasion chamber (BD Biosciences, San Jose, CA) according to the instructions provided by the manufacturer. The Ad-NT- or Ad-miR-143-infected cells (7.5 × 104/750 μl) were loaded on the transwell chamber (8 μm pore) covered with growth factor-reduced Matrigel. Ten nanogram per milliliter of EGF was placed in the lower chamber. The number of cells that migrated to the lower surface during a 24-h incubation period was determined by counting the cells in four places [18]. In this assay system, the pore filter was covered with a thin layer of Matrigel; therefore, the cells have to digest Matrigel in order to reach the lower chamber.

Animals

Female athymic nu/nu mice (Balb/c, 7 weeks old) were provided by the Animal Center of Central South University. The animals were housed socially (four mice per cage) in a room under standard lighting conditions and temperature. Water and food were provided ad libitum. All animal experiments were conducted under an approved protocol from the Central South University and performed in accordance with the animal care guidelines of the Chinese Council.

Tumor metastasis model

The orthotopic liver metastasis model was established as previously described [19]. Briefly, each animal was anesthetized with 2 % isoflurane in 100 % O2. Anesthesia was maintained using a snout cone. The Ad-NT or Ad-miR-143 (10 MOI)-infected Panc-1 cells (2 × 105) were suspended in 0.1 ml of saline and then slowly injected into the spleen to induce hepatic metastases. Each group consisted of 12 animals. Ten minutes after injection, a splenectomy was performed. The peritoneum and skin were then closed and the animals were returned to their cages for recovery. Animals were monitored daily. One additional injection of Ad-NT or Ad-miR-143 (1 × 108 pfu/100 μl saline) was given intraperitoneally at day 10 after the surgery. Animals were sacrificed by CO2 inhalation 25 days after inoculation. Livers were harvested and weighed.

Tumor growth study

Approximately 5 × 106 Panc-1 tumor cells were subcutaneously injected into the right hind limbs of mice. After the tumors had grown to about 8 mm in diameter, mice were randomly divided into two groups: Ad-NT or Ad-miR-143 injection group. Each group contains 12 animals. Viruses (1 × 108 pfu in 50 μl saline) were intratumorally injected once a week for 4 weeks. Tumors were measured in two dimensions every 2 days and tumor volume (V) was calculated using the following formula: \( V = \left( {1/2} \right){S^2} \times L \) (S, the shortest dimension; L, the longest dimension) [16]. Animals were euthanized when the subcutaneous tumor reached a size that required sacrifice (41 days after radiation).

Statistical analysis

Data were analyzed using Statistical Package for the Social Sciences, version 14.0 (Chicago, IL). Differences between groups were detected with two-tailed student t test. All data are presented as mean ± standard error of the mean. A p < 0.05 was considered statistically significant.

Results

miR-143 expression reduced mRNA expressions and protein levels of ARHGEF1, ARHGEF40, and KRAS genes

Panc-1 is a human pancreatic cancer cell line, expressing mutated KRAS proteins [20]. Previous studies demonstrated that K-RAS2 is a target of miR-143 [13, 14]. GEF1 and GEF40 genes were predicted to be the targets of miR-143. We, therefore, measured K-RAS2, GEF1, and GEF40 mRNA expression and protein levels in Ad-NT- and Ad-miR-143-infected Panc-1 cells. miR-143 expression (Fig. 1a) obviously reduced K-RAS2, GEF1, and GEF40 mRNA expression (Fig. 1b) and protein levels (Fig. 1c). The effect of AdsiNT on K-RAS2, GEF1, and GEF40 mRNA expression was tested in a separated experiment and no significant effect was observed (data not shown).
https://static-content.springer.com/image/art%3A10.1007%2Fs13277-012-0446-8/MediaObjects/13277_2012_446_Fig1_HTML.gif
Fig. 1

Inhibition of GEF1, GEF40, and K-RAS mRNA and protein expression by miR-143 expression. a Representative of Northern blots of miR-143 expression. Panc-1 cells were infected with Ad-NT or Ad-miR-143 virus for 24 h. Total RNA and microRNA were isolated as described in “Materials and methods.” Staining of 28 S and 18 S of total RNA was used as a reference of RNA intact and loading. b Representative of RT-PCR amplifications of GEF1, GEF40, and K-RAS mRNA expression. c Representative of Western blots of GEF1, GEF40, and K-RAS protein levels. d Percent densitometry values of GEF1, GEF40, and K-RAS protein levels. miR-143 expression obviously inhibited GEF1, GEF40, and K-RAS mRNA and protein expressions

miR-143 decreased Rho GTPase activity, MMP2, and MMP9 protein levels, and elevated E-cadherin protein level

The Rho GEFs activate Rho GTPases. RAS oncogene also regulates Rho GTPase activity [10]. We, therefore, proposed that decreased K-RAS, GEF1, and GEF40 gene expressions lead to inhibition in the constitutive activity of Rac1, Cdc42, and RhoA proteins. GTPase activity assays were performed as described in the “Materials and methods.”. Results revealed that miR-143 expression significantly inhibited the Rac1 (Fig. 2a), Cdc42 (Fig. 2b), and RhoA (Fig. 2c) activity. Previous studies revealed that Rho GTPases control the expression of MMPs [7]. MMP-2 and MMP-9 degrade type IV collagen, which is the major component of the basement membrane [21]. It was also revealed that Rho GTPases regulate the protein level of E-cadherin [22]. We further measured the protein levels of MMP-2, MMP-9, and E-cadherin in Panc-1 cells infected with Ad-NT or Ad-miR-143. miR-143 expression significantly decreased the protein levels of MMP-2 and MMP-9 but significantly increased the protein level of E-cadherin (Fig. 2d, e).
https://static-content.springer.com/image/art%3A10.1007%2Fs13277-012-0446-8/MediaObjects/13277_2012_446_Fig2_HTML.gif
Fig. 2

Rho GTPases activities and MMP-2, MMP-9, and E-cadherin protein expression. GTPase activation assays were performed with Panc-1 cells infected with Ad-NT or Ad-miR-143 virus for 24 h. The constitutively activated Rac-1 (a), Cdc42 (b), and RhoA (c) were subjected to the pull down assay and Western blot compared with total GTPase level in lysate. d Representative of Western blots of four independent experiments. e Percent densitometry values of MMP-2, MMP-9, and E-cadherin levels. *p < 0.001 for Rac-1, Cdc42, and RhoA activity, *p < 0.01 for E-cadherin and MMP-2 protein levels, *p < 0.05 for MMP-9 protein level. Data were presented as the means ± SEM of five separate experiments

miR-143 inhibited Panc-1 cell migration and invasion

The role of miR-143 on pancreatic cancer cell invasion and metastasis was tested in vitro by its effect on the migration and invasion of Panc-1 cells. Panc-1 cells were infected with Ad-miR-143 or Ad-NT control virus for 24 h and then subjected to migration (Fig. 3a) and invasion (Fig. 3b) assay with or without EGF stimulation. Consistent with previous studies, EGF significantly increased Panc-1 cell migration and invasion. miR-143 expression significantly inhibited the constitutive migration and invasion as well as EGF-induced migration and invasion in Panc-1 cells.
https://static-content.springer.com/image/art%3A10.1007%2Fs13277-012-0446-8/MediaObjects/13277_2012_446_Fig3_HTML.gif
Fig. 3

miR-143 expression inhibited migration and invasion of Panc-1 cells. a Ad-miR-143-mediated inhibition of migration. Panc-1 cells were infected with Ad-NT or Ad-miR-143 for 24 h. The cells were then loaded into the upper chamber and 10 ng/ml of EGF was placed in the lower chamber (EGF+) or not (EGF−). The results are expressed as migrated cell numbers in four places under microscopy at ×400 magnification. b Ad-miR-143-mediated inhibition of invasion. The cells were incubated for Matrigel invasion assay with or without 10 ng/ml of EGF. Data were presented as the means ± SEM of five separate experiments

miR-143 inhibited metastasis and growth of pancreatic cancer in animal models

To investigate the role of miR-143 on hepatic metastasis of pancreatic cancer, we used an orthotopic liver metastasis model in which Ad-miR-143-infected Panc-1 cells were injected into the spleens of Balb/c mice. To maintain the effectiveness of miR-143, one additional injection of Ad-miR-143 was given 10 days postsurgery. By day 25, after inoculation with Ad-NT-infected Panc-1 cells, mice developed hemorrhagic ascites and bulky liver metastases. In contrast, animals that were inoculated with Ad-miR-143-infected Panc-1 cells had very few grossly visible liver metastases (Fig. 4a). Quantitative analysis of liver weights (Fig. 4b) showed about 50 % weight reduction in mice inoculated with Ad-miR-143-infected Panc-1 cells compared to Ad-NT-infected Panc-1 cells-inoculated mice (P < 0.01). We also tested tumor growth inhibition of miR-143 in a xenograph animal model. Injection of Ad-miR-143 virus significantly inhibited tumor growth 19 days after first virus injection (Fig. 4c). To further validate whether tumor growth inhibition was caused by miR-143-regulated gene expression, we measured K-RAS2, GEF1, and GEF40 mRNA levels in tumor tissues. RT-PCR showed that the mRNA levels of K-RAS2, GEF1, and GEF40 genes were significantly decreased in tumor tissues from mice injected with Ad-miR-143 (Fig. 4d, e), suggesting that upregulation of miR-143 level can inhibit tumor growth of pancreatic cancer in vivo through downregulating K-RAS2, GEF1, and GEF40 expression.
https://static-content.springer.com/image/art%3A10.1007%2Fs13277-012-0446-8/MediaObjects/13277_2012_446_Fig4_HTML.gif
Fig. 4

Inhibition of liver metastasis and tumor growth. a Representative of liver metastases. Livers of mice inoculated with Ad-NT-infected Panc-1 cells had bulky metastases, whereas livers of mice inoculated with Ad-miR-143-infected Panc-1 cells had few visible metastases. b Liver weights. Data were given as mean ± SEM. *P < 0.01 vs. liver of mice inoculated with Ad-NT-infected cells. N = 12. c Tumor growth in xenograft Panc-1 tumor model. Ad-miR-143 significantly inhibited tumor growth from day 19 post-first virus injection (day 1). *p < 0.05, **p < 0.01, #p < 0.01 vs. Ad-miR-143-injected mice. N = 12. d Representative of RT-PCR amplification of GEF1, GEF40, and K-RAS genes. e Percent densitometry values of GEF1, GEF40, and K-RAS mRNA levels. miR-143 expression obviously inhibited GEF1, GEF40, and K-RAS mRNA expression. *p < 0.01, **p < 0.001 vs. Ad-siNT-injected mice

Discussion

Despite having a better understanding of the molecular events that govern the metastasis of pancreatic cancer than ever before, metastasis remains a clinical challenge. Identification of new biomarkers that play a central role in the metastasis of pancreatic cancer will benefit diagnosis and targeting therapy of the disease. In this study, we explored the effect of miR-143 on the metastasis of pancreatic cancer cells in vitro and in vivo. Restoration of miR-143 expression inhibited migration and invasion of Panc-1 cells, metastasis, and growth of pancreatic tumor. The tumor suppressive role of miR-143 is mediated through downregulating GEF1, GEF40, and K-RAS genes' expression; inhibiting RhoA, Rac1, and Cdc42 activity; and lowering MMP-2 and MMP-9 protein levels while elevating E-cadherin protein level. This study suggests that miR-143 is a key molecule involved in a multitude of processes related to pancreatic cancer metastasis.

miRNAs have been documented to function both as tumor suppressor genes and oncogenes. However, the regulatory role of miRNA is extraordinarily complex. Recent studies demonstrated that a single miRNA can regulate the expression of hundreds of genes, while a single gene can be regulated by multiple miRNAs [23]. It is widely reported that miRNAs are abnormally expressed in many types of cancers [11]. Loss of miR-143 expression has been reported in many cancer types, including pancreatic cancer, and restoration of miR143 expression has been shown to abrogate tumorigenesis [12, 13]. It has been revealed that the mutant K-RAS gene is a target of miR-143 [13]. Consistent with previous findings, miR-143 expression inhibited K-RAS-2 mRNA expression in Panc-1 cells in this study. Moreover, we experimentally identified two new targets of miR-143, the GEF1 and GEF40 genes. A recent study demonstrated that mutant K-RAS mediates migratory and invasive properties of colon cancer cells though regulating Rho GTPase activity [10]. In addition, GEFs commonly activate Rho GTPases through catalyzing the exchange of GDP for GTP [9]. As expected, miR-143-mediated inhibition of K-RAS, GEF1, and GEF40 genes' expression reduced constitutive RhoA, Rac1, and Cdc42 activity in this study.

The expression and activity of RhoA, Rac1, and Cdc42 have been previously observed to be upregulated in pancreatic tumors [9]. In this study, miR-143 expression significantly inhibited the activity of RhoA, Rac1, and Cdc42, suggesting that loss of miR-143 expression might be a key cause for the observed increase in Rho GTPases activity in pancreatic cancer. In addition, many studies have investigated the role of Rho family GTPases in tumor invasion and metastasis. It is widely observed that Rho GTPases tightly regulate the key steps of metastasis, including E-cadherin-mediated cell to cell interactions and MMP expression, which ultimately lead to the acquisition of an invasive phenotype [10]. In this study, miR-143 expression significantly increased E-cadherin expression and reduced MMP-2 and MMP-9 expressions. MMP-2 and MMP-9 degrade the major component of the basement membrane, which is a key step during the initial stages of invasion [21]. Loss of E-cadherin expression has been observed along with upregulation of MMP-2 and MMP-9 levels in highly metastatic undifferentiated carcinomas of the pancreas [8, 24]. In this study, we demonstrated that increased E-cadherin and decreased MMP-2 and MMP-9 expressions in response to increased miR-143 expression correlated with the inhibition of migration and invasion in Panc-1. Furthermore, miR-143 expression inhibited liver metastasis and xenograft tumor growth in animal models. Therefore, our study highlighted the key role of miR-143 in the Rho signaling pathway and metastasis of pancreatic cancer.

Besides the novel findings on the role of miR-143 in Rho signaling and regulation of tumor metastasis described above, this study also revealed that miR-143 is a potential target for targeting therapy of pancreatic cancer. In the Panc-1 xenograft tumor model, intratumoral injection of adenovirus-delivered miR-143 expression cassette significantly inhibited tumor growth. In the liver metastasis model, significant inhibition of liver metastasis was observed in mice inoculated with Ad-miR-143-infected Panc-1 cells. Therefore, our study suggests that miR-143 is an ideal target to develop specific agents for targeting therapy of pancreatic cancer.

In conclusion, loss of miR-143 expression might be responsible for the highly aggressive and metastatic characteristics of pancreatic cancer. Restoration of miR-143 expression can inhibit metastasis and invasion of pancreatic cancer cells. miR-143 may play a central role in the metastasis of pancreatic cancer by downregulating the expression of Rho GEF 1 and 40 as well as K-RAS gene expression, and subsequently inhibiting RhoA, Rac1, and Cdc42 activities. The Rho GTPases further mediate metastasis through their downstream molecules, such as E-cadherin and MMPs. Our study highlighted the key role of miR-143 in pancreatic cancer metastasis and suggested that miR-143 is an ideal target for pancreatic cancer therapy.

Acknowledgments

This work has been supported by the China National High-tech project (Grant nos. 2007AA021809 and 2007AA021803 to YC).

Conflicts of interest

None

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© International Society of Oncology and BioMarkers (ISOBM) 2012