hsa-miR-520h downregulates ABCG2 in pancreatic cancer cells to inhibit migration, invasion, and side populations

Background: Expression of ABCG2 is normally absent or low in the pancreas, but high in human pancreatic cancer cells. The mechanism by which ABCG2 is altered in human cancers remains unknown. Methods: We investigated ABCG2 expression in four pancreatic cancer cell lines, and used three microRNA (miRNA) target prediction programmes, and information from the existing literature to predict and identify hsa-miR-520h as an miRNA that targets ABCG2. The function of this miRNA was investigated by transient transfection of the pancreatic cancer cell line PANC-1 with oligonucleotides that mimic hsa-miR-520h. Results: Results showed that both mRNA and protein levels of ABCG2 were reduced, indicating that it was a target of hsa-miR-520h. Introduction of hsa-miR-520h mimics into PANC-1 cells also resulted in inhibition of cell migration and invasion, and reduction of side population cells. Cell proliferation, cell cycle progression and apoptosis were not affected. Conclusions: We propose that the effects of hsa-miR-520h may be, at least in part, caused by its regulation of ABCG2. Thus, our findings provide a new insight into the function of miRNA in the regulation of ABCG2 expression in pancreatic cancer. Gene therapy using miRNA mimics may therefore be useful as a pancreatic cancer therapy.

Pancreatic cancer is the fourth leading cause of cancer-related death in Western countries and has the lowest patient survival rate of any solid cancer (Jemal et al, 2009). One of the main reasons for its mortality is that pancreatic cancer cells have a high potential for invasion and metastasis. Although invasion and migration are the overwhelming causes of mortality in patients with solid tumours, our understanding of their molecular determinants is limited (Fidler, 2003;Weigelt et al, 2005;Gupta and Massagué, 2006). Studies on tumour invasion and metastasis have revealed a critical role for microRNAs (miRNAs), which are small, endogenous noncoding RNAs that post-transcriptionally regulate a variety of genes pivotal for invasion or metastasis (Lim et al, 2005;Dalmay and Edwards, 2006;Kim et al, 2008).
In this study, on the basis of previous reports To et al, 2008b), we investigated whether hsa-miR-520h targets ABCG2 mRNA. We also investigated the role of hsa-miR-520h in proliferation, cell cycle progression, apoptosis, migration, and invasion in PANC-1 cells. As side-population cells of pancreatic cancer that overexpress ABCG2 have been identified as promoting carcinogenesis (Ho et al, 2007;Shi et al, 2008;Dou et al, 2009;, we also investigated the effects of hsa-miR-520h on these cells. We determined whether the effects of hsa-miR-520h are linked to downregulation of ABCG2 expression. On the basis of our findings, we propose a model explaining how loss of hsa-miR-520h expression and subsequent activation of ABCG2 expression are critical events in the invasion and migration of human pancreatic cancer cells.

Cell proliferation, cell cycle, and apoptosis analysis
Cell proliferation was determined using an MTT assay. PANC-1 cells were seeded in 96-well culture plates (Costar, Cambridge) at a density of 1 Â 10 3 cells per well, transfected with the indicated miRNA and incubated for 24 h before adding 10 ml of MTT reagent to each well and incubating at 371C for 4 h; thereafter, the medium was replaced with 150 ml dimethyl sulphoxide (DMSO) and oscillated for 10 min. Viable cells were measured by absorbance at 490 nm wavelength using a microplate reader. Cell cycle and apoptosis were assessed by flow cytometry (Becton Dickinson, San Jose, CA, USA). PANC-1 cells were grown and transfected as above. For cell cycle analysis, cells were collected, washed with phosphate-buffered saline (PBS) and fixed in ethanol at À201C overnight before being collected by centrifugation, washed with PBS, and resuspended in 500 ml of PBS with 0.2% Triton X-100, 10 mM EDTA, 100 mg ml À1 RNase A, and 50 mg ml À1 propidium iodide (PI) at room temperature for 30 min. For cell apoptosis, cells were collected, washed with PBS, suspended in 100 ml 1 Â binding buffer, stained with 5 ml annexinfluorescein isothiocyanate (FITC), and 5 ml PI at room temperature for 15 min in the dark.

Cell migration and invasion analysis
Cell migration and invasion were assayed using a chamber of 6.5 mm in diameter, with 8 mm pore size (Corning, Corning, NY, USA). At 24 h post-transfection, PANC-1 cells were added to the upper chamber, which was coated with 1 mg ml À1 matrigel for invasion assays, and 0.6 ml of 10% FBS-DMEM was added to the lower chamber. Cells were incubated for 24 h at 371C, and nonmigrating or non-invading cells were removed with cotton swabs. 10 0 10 4 10 3 10 2 10 1 10 0 10 4 10 3 10 2 10 1 10 0 10 4 10 3 10 2 10 1 10 0   Cells migrating to or invading the bottom of the membrane were stained with 0.1% crystal violet for 30 min at 371C, and washed with PBS. Stained cells were soaked in 33% ice-cold acetic acid and oscillated for 10 min. Cell migration or invasion was assessed by absorbance at 570 nm wavelength using a microplate reader (Tecan, Shanghai, China).

Side-population cell analysis
For analysis of side-population cells, PANC-1 cells were transfected with miRNA for 24 h, then incubated with Hoechst 33342 (5 mmol ml À1 , Sigma) for 90 min at 371C, either alone or in combination with 50 mmol per ml verapamil. Cells were analysed in a FACS Vantage cell sorter (Becton Dickinson) using dualwavelength analysis (blue 425 nm, red 675 nm) after excitation with 350 nm UV light. Propidium iodide-positive cells were excluded.

Statistical analysis
Each experiment was conducted at least three times. All values were reported as mean ± s.d. Differences between experimental groups and controls were assessed by Student's t-test using Excel software. Po0.05 was considered statistically significant.

Pancreatic cancer cell lines expressed variable levels of ABCG2
Recent studies have revealed that ABCG2 is overexpressed in many human cancers. Therefore, we checked the expression of ABCG2 in four human malignant pancreatic cancer cell lines: BxPC-3, CFPAC-1, PANC-1, and SW1990. BxPC-3 and CFPAC-1 are lowgrade cancer cell lines, and PANC-1 and SW1990 are high-grade lines. Western blot analysis showed that ABCG2 was overexpressed in PANC-1 and SW1990 cells, and underexpressed in BxPC-3 and CFPAC-1 cells ( Figure 1A). These results were confirmed by qRT -PCR analysis ( Figure 1B).

hsa-miR-520h targets ABCG2
Using miRNA target predicting programmes and previous reports, we hypothesised that the miRNA hsa-miR-520 h targeted ABCG2, and we chose PANC-1 cells to test our hypothesis. To verify our predicted target of hsa-miR-520h, we transfected PANC-1 cells with hsa-miR-520h mimics (miR-520h), negative control miRNA mimics (NC), and blank control culture medium (mock). Western blot analysis showed that, at 24 h post-transfection, overexpression of hsa-miR-520h resulted in a significant decrease of endogenous ABCG2 protein levels, compared with cells transformed with NC or hsa-miR-520h downregulates ABCG2 mock ( Figure 2A). Inhibition of expression by miRNAs mediates mRNA levels; hence we used qRT -PCR to examine ABCG2's mRNA levels. Overexpression of hsa-miR-520h in PANC-1 cells led to a corresponding decrease of endogenous ABCG2 mRNA at 24 h post-transfection ( Figure 2B). These observations suggested that hsa-miR-520h downregulates ABCG2 expression by inhibiting translation or causing mRNA instability. Taken together, these data indicate that hsa-miR-520h targets ABCG2.
Effects of hsa-miR-520h on proliferation, cell cycle, and apoptosis To investigate whether hsa-miR-520h influenced cell proliferation, cell cycle, or apoptosis of PANC-1 cells by inhibiting translation of the indicated target ABCG2 mRNA, we transiently transfected PANC-1 cells with hsa-miR-520h mimics for 24 h. Ectopic expression of hsa-miR-520h had no significant inhibition on cell proliferation compared with that of control groups ( Figure 3A). The cell cycle distribution of transfected cells was examined by flow cytometry, with no significant G0/G1 accumulation phenotype in PANC-1 cells transfected with hsa-miR-520h (56.43%) compared with NC (55.34%) or mock (49.68%) control-transfected cells ( Figure 3B). We also examined the cell apoptosis of transfected cells by flow cytometry, and found no significant increase in apoptosis in PANC-1 cells transfected with hsa-miR-520h (7.31%) compared with NC (6.45%) or mock (5.70%) transfected cells ( Figure 3C).
hsa-miR-520h inhibits migration and invasion of PANC-1 cells To determine whether hsa-miR-520h and its target gene are associated with tumour migration and invasion, we transfected PANC-1 cells with hsa-miR-520h mimics or NC, or subjected them to mock transfection, and evaluated cell migration and invasion. As shown in Figure 4A and B, the migration ability of hsa-miR-520h mimic-transfected cells was significantly decreased by 55%, compared with that of controls. A matrigel invasion assay was performed in parallel to determine the effect of transfection with hsa-miR-520h mimics. As shown in Figure 4A and C, the invasion ability of hsa-miR-520h-transfected cells was significantly decreased by 53%, compared with that of controls. Taken together, these results suggested that hsa-miR-520h functions as a potent suppressor of PANC-1 cell migration and invasion through downregulation of ABCG2 expression.
hsa-miR-520h depletes the side population of PANC-1 cells Previous studies suggested that ABCG2 has an important role in the identity of side-population cells. Therefore, we examined the influence of hsa-miR-520h on the population of tumour cells that exclude Hoechst 33342 dye, a strategy that is used to identify side-population cells. Flow cytometry of hsa-miR-520h-transfected PANC-1 cells showed that the side population was 1.51%, compared with 2.79% in NC-transfected and 3.06% in mock-transfected cells ( Figure 5). These data indicated that   0  1000  800  600  400  200  0   1000  800  600  400  200  0  1000  800  600  400  200  0  1000  800  600  400  200  0   1000  800  600  400  200  0  1000  800  600  400 200 0 Figure 5 Overexpression of hsa-miR-520h decreases the side-population compartment of PANC-1 cells. Side-population cells of PANC-1 cells transfected with hsa-miR-520h account for 1.51% of the total number of cells, compared with 2.79% for NC-treated, or 3.06% for mock-treated, as measured by FACS analysis. hsa-miR-520h downregulates ABCG2 hsa-miR-520 h has a critical role in the identification of the side population, through downregulation of ABCG2 expression.

DISCUSSION
To investigate the interaction between miRNAs and their target genes in pancreatic cancer, we determined the expression levels of ABCG2 in four pancreatic cancer cell lines. Both western blot and qRT -PCR analysis showed that the cell lines expressed variable levels of ABCG2, with high expression in PANC-1 and SW1990 cells, and low expression in BxPC-3 and CFPAC-1 cells (Figure 1). We used miRNA target prediction programmes and previous studies to find an miRNA that potentially regulated ABCG2, and selected PANC-1 cells for verification using transfected miRNA mimics. In the transfected cells, ABCG2 was downregulated at both the mRNA and protein levels by the proposed target miRNA, hsa-miR-520h ( Figure 2). These data suggested that ABCG2 is a target of hsa-miR-520h in pancreatic cells.
Overexpression of ABCG2 promotes migration and invasion in a variety of different tumour types (Mohan et al, 2006;Ho et al, 2007;Chiou et al, 2008;Okamoto et al, 2009). The mechanism by which ABCG2 becomes overexpressed is not entirely clear, but a possible mechanism is through miRNA regulation. MicroRNAs can function as tumour suppressors, oncogenes, or as promoters or suppressors of metastasis. We transfected hsa-miR-520h into PANC-1 cells and observed cell proliferation, cycle, apoptosis, migration, and invasion. Introduction of hsa-miR-520h resulted in a significant decrease in cell migration and invasion, but with no significant effects on cell proliferation, cell cycle distribution, or apoptosis (Figures 3 and 4). This is the first study to show that hsa-miR-520h inhibits migration and invasion in pancreatic cancer cells, through a possible mechanism of negative regulation of ABCG2 expression.
In this study, FACS analysis at 24 h after transfection of PANC-1 cells with miRNA mimics showed a remarkable decrease in the proportion of side-population cells ( Figure 5). Therefore, we propose that hsa-miR-520h may have a critical role in carcinogenesis by way of affecting side-population cells by influencing ABCG2 expression. However, Patrawala et al (2005) reported that ABCG2-positive and -negative tumour cells have similar tumourigenicity; hence, the precise reasons why ABCG2 is associated with pancreatic cancer cells require further study.
In conclusion, we show that hsa-miR-520h has a key role not only in inhibiting migration and invasion, but also in decreasing the proportion of side-population cells of PANC-1. The possible mechanism of these effects may be through downregulation of ABCG2 expression. As no effective approaches for curing pancreatic cancer exist at present, we propose that gene therapy targeting hsa-miR-520h/ABCG2 should be investigated further as a potential alternative therapeutic strategy for pancreatic cancer.