Human tumor tissue samples and adjacent noncancerous controls were obtained by surgical resection from six patients with pancreatic cancer, at the Department of General Surgery, First People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China. All samples were derived from patients who had not received adjuvant treatment including radiotherapy or chemotherapy prior to surgery. All samples were snap-frozen and stored in liquid nitrogen after collection. Written informed consent was obtained from all subjects, and the study was approved and supervised by the Ethics Committee of the First People’s Hospital, School of Medicine, Shanghai Jiao Tong University.

HPDE, pancreatic cancer cells BxPC-3, CFPAC-1, CAPAN-1, SW1990, AsPC-1, PANC-1 and HS-766T were all obtained from Chinese Academy of Sciences Cell Bank (Shanghai, China). PANC-1 and HS-766T were grown in 5% CO₂ saturated humidity, at 37°C, and cultured in DMEM supplemented with 2 mmol/l glutamine and 10% fetal bovine serum (FBS) (both from Gibco, USA) and subcultured by harvesting with trypsin-EDTA. HPDE, BxPC-3, CAPAN-1, AsPC-1, CFPAC-1 and SW1990 cells were cultured in RPMI-1640 (Gibco) supplemented with 10% FBS.

Total RNA was isolated from the cultured cells and tissue samples by using an RNA isolation kit (Takara Bio, Inc.) according to the manufacturer’s instructions. Reverse transcription and RT-qPCR kits (Takara Bio, Inc.) were applied to evaluate expression of MALAT-1. Primers used were: MALAT-1 forward, 5′-GAATTGCGTCATTTAAAGCCTAGTT-3′ and reverse, 5′-GTTTCATCCTACCACTCCCAATTAAT-3′; GAPDH forward, 5′-ACAGTCAGCCGCATCTTCTT-3′ and reverse, 5′-GACAAGCTTCCCGTTCTCAG-3′. The expression of GAPDH was detected as the endogenous control. Relative mRNA expression of MALAT-1 was calculated with the comparative threshold cycle (Ct) (2^−ΔΔCt) method.

Cells were lysed in RIPA lysis buffer and the protein concentration was determined (Beyotime). Total proteins were fractionated using SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. The membranes were blocked in 3% bovine serum albumin in TBST buffer containing 0.1% Tween-20 and then incubated with the indicated primary antibodies at 4°C overnight. Appropriate secondary antibodies were incubated at room temperature for 1 h and detected using the enhanced chemiluminescence detection system. The data was adjusted against loading control using β-actin. The antibodies used for western blot analyses were: mouse anti-N-cadherin, mouse anti-E-cadherin (BD Biosciences, Bedford, MA, USA); mouse anti-vimentin, rabbit anti-ALDH (Abcam); rabbit anti-Slug, rabbit anti-Snail, mouse anti-CDC2, rabbit anti-matrix metalloproteinases-2 (MMP-2), rabbit anti-MMP-9, rabbit anti-PCNA (Cell Signaling Technology); mouse anti-p21, mouse anti-p53, mouse anti-CD44, rabbit anti-CD24, mouse anti-β-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Based on principles of shRNA design and the human MALAT-1 structure (NR_002819), three plasmid vectors encoding shRNA directed against MALAT-1 mRNA were constructed. The three shRNA targeting sequences were: shRNA-M1, 5′-GAGTAACTGGCATGTGAGCAA-3′; shRNA-M2, 5′-CATGACGGAGGTTGAGATGAA-3′; shRNA-M3, 5′-AAGCCGAAATAAATGAGAGAT-3′. The scrambled sequence, 5′-TTC′TCCGAACGTGTCACGT-3′, was used as a negative control that does not target any known human mRNA. The preparation of lentivirus expressing human MALAT-1 shRNA was performed using the GV-248 lentiviral RNAi expression system (Genechem, Shanghai, China). AsPC-1 and CFPAC-1 were infected with lentiviral particles containing specific or negative control vectors, and the polyclonal cells with puromycin resistance were selected for further experiments.

Briefly, ~1,000 cells were added to each well of a 6-well culture plate. After 2 weeks of incubation, cell colonies were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min and then stained with crystal violet for 30 min. Individual clones with >50 cells were counted.

A real-time imaging system (IncuCyte™) was used to measure cell proliferation using non-label cell monolayer confluence approach. IncuCyte provides the ability to acquire high quality, phase-contrast images and an integrated confluence metric as a surrogate for cell number (5). We used a similar approach to determine the effect of MALAT-1 on pancreatic cancer cell proliferation. Cell confluence was compared between M-nc and M-si1 stable transfected cancer cells.

Cells were cultured and grown to 100% confluence. Wounds were scratched in the monolayer with a 100-μl pipette tip. Suspended cells and debris were washed with PBS buffer, then the cells were incubated in RPMI-1640 medium containing 2% FBS at 37°C. The migration of cells into the wounded areas was evaluated at the indicated times using an inverted microscope, and then photographed. Three different areas in each assay were selected to measure the distance of the migrating cells to the origin of the wound.

The cell invasive and migratory potential were evaluated using Boyden chamber and Transwell assay, respectively. Briefly, Boyden chamber was conducted using specialized MilliCell chambers, which included a 24-well tissue culture plate with 12 cell culture inserts (Millipore, Bedford, MA, USA). The inserts contained an 8 μm pore size polycarbonate membrane with a pre-coated thin layer of Matrigel (BD Biosciences). Ten percent FBS-containing medium was placed in the lower chambers to act as a chemo-attractant. Then, 1×10⁵ AsPC-1 and CFPAC-1 in a 100 μl volume of serum-free medium were placed in the upper chambers and incubated at 37°C for 48 h. Invasive cells on the lower surface of the membrane, which had invaded the Matrigel and had migrated through the polycarbonate membrane, were stained by the staining solution, and counted under a microscope in five randomly selected fields at a magnification of ×200. Transwell assay was the same as the Boyden chamber with the exception that no Matrigel was used and the permeating time for cells was 24 h.

For analysis of the cell apoptosis, cells were harvested at 70–80% confluence and incubated with reagent containing Annexin V-FITC and propidium iodide (BD Biosciences) for 15 min in darkness at room temperature. Apoptotic cells were analyzed using FACSCalibur flow cytometer (BD Biosciences). For cell cycle analysis, cells were fixed in 70% ethanol at 4°C overnight and then treated with RNase A (50 μg/ml) and stained with propidium iodide (25 μg/ml) for 30 min at 37°C. Distribution of cell-cycle phases was determined using ModFit software (BD Biosciences).

All statistical analyses were performed using SPSS 13.0. The data are presented as means ± SEM from at least three separate experiments. The difference between two groups was analyzed by the Student’s t-test (two-tailed). In vitro cell growth assay was tested using factorial design one-way analysis of variance (ANOVA). The P-value of <0.05 was considered to indicate a statistically significant difference.

To assess the role of MALAT-1 in pancreatic cancer, we first assayed MALAT-1 expression in tumor tissue samples and adjacent non-cancerous pancreas tissues from six patients with pancreatic cancer. The results showed that MALAT-1 expression was significantly higher in five cases of tumor tissues than in matched paracancerous controls (Fig. 1A). Furthermore, we compared the expression of MALAT-1 between pancreatic cancer cell lines and normal HPDE cells. Notably, consistently higher expression level of MALAT-1 was found in all seven pancreatic cancer cell lines relative to the HPDE, among which five were upregulated >4-fold (Fig. 1B). Taken together, it is suggested that upregulation of MALAT-1 is a frequent event in human pancreatic cancer.

Among the pancreatic cancer cell lines, AsPC-1 and CFPAC-1 had relatively higher expression levels of MALAT-1 (Fig. 1B), therefore these two cell lines were chosen to study the functions of endogenous MALAT-1 through a loss-of-function approach. We used a lentiviral shRNA vector to specifically and stably knock down the expression of MALAT-1 in AsPC-1 and CFPAC-1. A knockdown effect was observed by RT-qPCR, and we found that M-si1 achieved the greatest efficacy in silencing MALAT-1 expression compared to the negative control M-nc (Fig. 2A). Thus, stable transfection cancer cell lines AsPC-1/M-nc and AsPC-1/M-si1, as well as CFPAC-1/M-nc and CFPAC-1/M-si1 were used in the following experiments.

Subsequently, we examined the effect of decreased MALAT-1 expression on pancreatic cancer cell proliferation in vitro by the IncuCyte real-time video imaging system. The determined growth curves showed that suppressing MALAT-1 significantly reduced cell proliferation in comparison with M-nc cells (Fig. 2B). The analysis of plate clone formation assay in CFPAC-1 was concordant with the above result (Fig. 2C). However, for AsPC-1, the two groups had no cell clone formation. Furthermore, western blot analysis showed that, compared to M-nc, the level of proliferation marker PCNA was decreased in the M-si1 groups (Fig. 2D).

Next, to investigate the mechanism involved in growth suppression, we carried out flow cytometry analysis. The cell cycle results showed that the G₂/M-phase fraction increased from 14.0±0.9% (AsPC-1/M-nc) to 23.9±0.7% (AsPC-1/M-si1), 1.8±0.2% (CFPAC-1/M-nc) to 4.6±0.7% (CFPAC-1/M-si1) respectively, indicating G₂/M cell cycle arrest after MALAT-1 knockdown (Fig. 3A). In addition, we detected cell apoptosis after MALAT-1 downregulation. Compared to the M-nc, significantly enhanced cell apoptosis was observed in the M-si1 group (AsPC-1/M-si1 6.46% vs. AsPC-1/M-nc 3.45%; CPFAC-1/M-si1 15.59% vs. CPFAC-1/M-nc 5.55%) (Fig. 3B).

The G₂ checkpoint prevents cells from entering mitosis when DNA is damaged, providing an opportunity for repair and stopping the proliferation of damaged cells. CDC2, the cyclin-dependent kinase that normally drives cells into mitosis, is the ultimate target of pathways that mediate rapid arrest in G₂/M (6). A previous study revealed that p53 plays an important role in maintaining G₂/M arrest and induces cell apoptosis (7). The contribution of p53 to G₂/M arrest and cell apoptosis involves some of its transcriptional targets. p21, a well-known cell cycle regulator, is a major target of p53 (7). Elevated expression of p21 may result in increased association with CDKs and inhibit their activity, blocking the entry of cells at the G₂-M-phase transition checkpoint, and induce apoptosis (8). Our results revealed that p21 and p53 protein levels significantly increased following MALAT-1 downregulation, while CDC2 expression decreased (Fig. 3C). Taken together, these results indicated an inhibiting effect of reduced MALAT-1 expression on pancreatic cancer cell growth possibly through inducing G₂/M cell cycle arrest and promoting cell apoptosis.

To examine the effect of MALAT-1 on pancreatic cancer cell migration, we used a wound-healing assay and a Transwell migration assay. According to the wound-healing assay, the migratory areas of AsPC-1/M-si1 and CFPAC-1/M-si1 were significantly smaller than those of control cells (Fig. 4A). Similarly, Transwell migration assay showed that, after 24-h incubation, the number of migrated cells in the M-si1 groups was significantly lower than that in the M-nc cells (AsPC-1/M-si1 45±4 vs. AsPC-1/M-nc 107±8; CPFAC-1/M-si1 79±8 vs. CPFAC-1/M-nc 154±12), indicating a decreased migratory ability following MALAT-1 downregulation (Fig. 4B). Next, using a Boyden chamber pre-coated with Matrigel, we determined changes in cell invasiveness after 48 h incubation. Compared with the M-nc cells, M-si1 cells revealed significantly decreased invasiveness (AsPC-1/M-si1 27±2 vs. AsPC-1/M-nc 82±5; CPFAC-1/M-si1 18±2 vs. CPFAC-1/M-nc 90±3) (Fig. 4C). Based on these results, knockdown of MALAT-1 clearly inhibited the migration and invasion of pancreatic cancer cells in vitro.

It is believed that acquiring the migratory characteristics of a mesenchymal-like state enhances the invasive capabilities of cancer cells. The EMT switch is related to an unfavorable prognosis in pancreatic ductal adenocarcinoma (9). During EMT of in situ tumor cells, expression of proteins that promote cell-cell contact such as E-cadherin can be lost, and mesenchymal markers such as vimentin, N-cadherin and the metalloproteinases MMP-2 and MMP-9 can be acquired, resulting in enhanced ability for cell migration and invasion (10). We therefore examined the expression of EMT-related genes in response to the knockdown of MALAT-1. The results showed that the level of E-cadherin was upregulated with the decrease in the level of MALAT-1, while N-cadherin and vimentin decreased (Fig. 5A). In addition, EMT-related transcriptional factors including Snail and Slug expression were also reduced (Fig. 5A). Moreover, we found that suppressing MALAT-1 expression decreased expression of MMP-2 and MMP-9 (Fig. 5A). Furthermore, CFPAC-1 cells were morphologically mixed populations of epithelial and spindle-shaped mesenchymal type cells in nature and led to typical epithelial morphology transition after MALAT-1 downregulation (Fig. 5B). However, the change of morphological features for MALAT-1 knockdown in AsPC-1 was not observed. Collectively, these findings suggest that knockdown of MALAT-1 inhibits pancreatic cancer cell migration and invasion possibly through suppression of EMT.

Accumulating evidence suggests that cells can acquire stem-like properties during induction of EMT (11). Pancreatic cancer stem cells (CSCs) have been identified based on the expression of CD24, CD44, and epithelial-specific antigen (ESA) (12). The expression of aldehyde dehydrogenase (ALDH) was also used as special markers of pancreatic CSCs (13). In order to gain insight into the role of MALAT-1 involved in regulating pancreatic CSCs, we performed western blot analysis to analyze the altered expression of pancreatic CSC-associated markers following MALAT-1 downregulation, and the results showed that the levels of CD44, CD24 and ALDH were significantly decreased in M-si1 compared with the M-nc groups (Fig. 5A). The above data suggest that MALAT-1 facilitates pancreatic cancer cell progression possibly through acquiring cancer stem-like properties.