MicroRNA-221 Mediates the Effects of PDGF-BB on Migration, Proliferation, and the Epithelial-Mesenchymal Transition in Pancreatic Cancer Cells

<div><p>The platelet-derived growth factor (PDGF) signaling pathway has been found to play important roles in the development and progression of human cancers by regulating the processes of cell proliferation, apoptosis, migration, invasion, metastasis, and the acquisition of the epithelial-mesenchymal transition (EMT) phenotype. Moreover, PDGF signaling has also been found to alter the expression profile of miRNAs, leading to the reversal of EMT phenotype. Although the role of miRNAs in cancer has been documented, there are very few studies documenting the cellular consequences of targeted re-expression of specific miRNAs. Therefore, we investigated whether the treatment of human pancreatic cancer cells with PDGF could alter the expression profile of miRNAs, and we also assessed the cellular consequences. Our study demonstrates that miR-221 is essential for the PDGF-mediated EMT phenotype, migration, and growth of pancreatic cancer cells. Down-regulation of TRPS1 by miR-221 is critical for PDGF-mediated acquisition of the EMT phenotype. Additionally, the PDGF-dependent increase in cell proliferation appears to be mediated by inhibition of a specific target of miR-221 and down-regulation of p27Kip1.</p></div

miR-221 is critical for the PDGF-mediated epithelial-mesenchymal transition phenotype, migration, and growth of AsPC-1 cells.

<p>AsPC-1 cells were transfected with a negative control, the miR-221 mimic, or antisense oligonucleotides to miR-221 (anti-mIR-221). The cells were then treated with PDGF-BB (20 ng/ml) for 24 h and subjected to qRT-PCR (A, C) or Western blot analysis (B) of the transcription factors and EMT-specific gene markers. AsPC-1 cells were transfected with a negative control, the miR-221 mimic, or antisense oligonucleotides to miR-221 (anti-mIR-221) and subjected to the Matrigel transmembrane invasion assay (D) in the presence or absence of 20 ng/ml PDGF-BB. AsPC-1 cells were transfected with a negative control, the miR-221 mimic, or antisense oligonucleotides to miR-221 (anti-mIR-221), followed by PDGF-BB treatment for 24 h, and then were stained with a FITC-conjugated antibody against the proliferation marker PCNA and DAPI. In total, 150 cells were counted per condition, and the percentage of PCNA-positive cells (E) is presented. All treatment experiments in this figure were carried out in triplicate, and the results are displayed as the means ± SD.</p

PDGF promoted cell migration and proliferation and altered the EMT phenotype in AsPC-1 cells.

<p>AsPC-1 cells incubated with vehicle or PDGF-BB (20 ng/ml) were subjected to the scratch wound assay (A) and Matrigel transmembrane invasion assay (B). The results are the mean±SD. of triplicate measurements of three independent experiments. AsPC-1 cells incubated with vehicle or PDGF-BB (20 ng/ml) for 24 h were subjected to the MTT cell proliferation assay (The results are indicated as the absorbance readings at 490 nm) (C) or stained with a FITC-conjugated antibody against the proliferation marker PCNA and DAPI (150 cells were counted per condition, and the percentage of PCNA-positive cells is presented.) (D). The results are the mean±SD. for triplicate assays of three independent experiments. Real time-PCR was used to determine the mRNA levels to evaluate the upregulated expression of transcription factors (E) and EMT-specific genes (F) in AsPC-1 cells incubated with vehicle or PDGF-BB (20 ng/ml, 24 hr). The relative mRNA levels were normalized to GAPDH. All of the treatments in this figure were carried out in triplicate, and the results are displayed as the means ± SD. G: Photomicrographs of AsPC-1 cells incubated with vehicle or PDGF-BB (20 ng/ml, 24 hr). Original magnification, 200X.</p

miR-221 is regulated by the PDGF-BB signaling pathway.

<p>A: The relative miRNA levels in AsPC-1 cells treated with vehicle or PDGF-BB (20 ng/ml, 24 hr) B, C: Time-course expression of the relative expression of miR-221-3p, miR-221-5p, miR-222-3p and miR-222-5p (B), and miR-221 transcripts (Pri-miR-221), Pre-miR-221 or mature miR-221 (C) normalized to GAPDH (for Pri-miR-221 or Pre-miR-221), or U6 small nuclear RNA (for miR-221-3p, miR-221-5p, miR-222-3p, miR-222-5p and mature miR-221) in AsPC-1 cells treated with vehicle or PDGF-BB (20 ng/ml, 24 hr). D: AsPC-1 cells were treated with vehicle or PDGF-BB (20 ng/ml), or transfected with a negative control, the miR-221 mimic, anti-mIR-221, or anti-mIR-221 and followed by PDGF-BB treatment., and subjected to qRT-PCR of miR221. All of the treatments in this figure were carried out in triplicate, and the results are displayed as the means ± SD.</p

PDGF-mediated inhibition of p27Kip1 by miR-221 Promotes Cell proliferation.

<p>(A, B): AsPC-1 cells were transfected with a negative control mimic, the miR-221 mimic, antisense oligonucleotides to miR-221 (anti-mIR-221) or p27Kip1 siRNA. The cells were then treated with PDGF-BB (20 ng/ml) for 24 h and subjected to qRT-PCR (A) and Western blot analysis (B) for p27Kip1. (C): AsPC-1 cells were transfected with negative control or p27Kip1 siRNA and subjected to the MTT cell proliferation assay (The results are indicated as the absorbance readings at 490 nm). (C) (D, E): AsPC-1 cells were transfected with a negative control mimic, the miR-221 mimic, antisense oligonucleotides to miR-221 (anti-mIR-221) or p27Kip1 siRNA. The cells were then treated with PDGF-BB (20 ng/ml) for 24 h and subjected to staining with a FITC-conjugated antibody against the proliferation marker PCNA and DAPI (E). In total, 150 cells were counted per condition, and the percentage of PCNA-positive cells is presented (D). All experiments in this figure were carried out in triplicate, and the results are displayed as the means ± SD.</p

PDGF mediated the EMT by miR-221 targeting TRPS1.

<p>AsPC-1 cells were transfected with a negative control mimic, the miR-221 mimic, antisense oligonucleotides to miR-221 (anti-mIR-221) or TRPS1 siRNA. The cells were then treated with PDGF-BB (20 ng/ml) for 24 h and subjected to qRT-PCR of TRPS1. AsPC-1 cells were transfected with a negative control mimic, the miR-221 mimic, antisense oligonucleotides to miR-221 (anti-mIR-221) or TRPS1 siRNA for 3 days and subjected to Western blot analysis of TRPS1, ZEB2, E-cadherin and vimentin. AsPC-1 cells were transfected with antisense oligonucleotides to miR-221 (anti-mIR-221). The cells were then treated with a negative control or miR-221 mimic for 3 days and subjected to qRT-PCR for ZEB2 (C), E-cadherin (D) and vimentin (E). AsPC-1 cells were transfected with a negative control mimic, the miR-221 mimic or antisense oligonucleotides to miR-221 (anti-mIR-221). The cells were then treated with PDGF-BB (20 ng/ml) for 24 h and subjected to immunofluorescence staining for vimentin (F) and E-cadherin (G) All treatments in this figure were carried out in triplicate, and the results are displayed as the means ± SD.</p

Gemcitabine treatment induces endoplasmic reticular (ER) stress and subsequently upregulates urokinase plasminogen activator (uPA) to block mitochondrial-dependent apoptosis in Panc-1 cancer stem-like cells (CSCs)

<div><p>Background</p><p>Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor survival rates. The presence of cancer stem-like cells (CSCs) is believed to be among the underlying reasons for the aggressiveness of PDAC, which contributes to chemoresistance and recurrence. However, the mechanisms that induce chemoresistance and inhibit apoptosis remain largely unknown.</p><p>Methods</p><p>We used serum-free medium to enrich CSCs from panc-1 human pancreatic cancer cells and performed sphere formation testing, flow cytometry, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and semi-quantitative western blotting to confirm the stemness of panc-1 CSCs. Hallmarks of endoplasmic reticulum (ER) stress, including IRE1, PERK, ATF4, ATF6α, GRP78 and uPA expression, were detected after gemcitabine treatment. Effects of gemcitabine-induced uPA expression on cell invasion, sphere formation, colony formation and gemcitabine sensitivity were detected. Electrophoretic mobility shift assays (EMSAs) and RNA-immunoprecipitation (RIP) were performed to detect interaction between the uPA mRNA 3’-UTR and mutant p53-R273H expressed by panc-1 CSCs. The effects of upregulated uPA by gemcitabine on apoptosis were detected by Annexin V-FITC/PI staining, and the impact of uPA on small molecule CP-31398-restored mutant p53 transcriptional activity was measured by a luciferase reporter assay.</p><p>Results</p><p>Enriched panc-1 CSCs expressing high levels of CD44 and CD133 also produced significantly higher amounts of Oct4 and Nanog. Compared with panc-1 cells, panc-1 CSCs presented chemoresistance to gemcitabine. ER stress gene detections demonstrated effects of gemcitabine-induced ER stress on both the pro-apoptotic and pro-survival branches. ER stress-induced ATF6α upregulated level of uPA by transcriptionally activating GRP78. Gemcitabine-induced uPA promoted invasion, sphere formation and colony formation and attenuated apoptosis induced by gemcitabine in panc-1 CSCs, depending on interaction with mutant p53-R273H. Upregulation of uPA abolished CP-31398-mediated restoration of mutant p53 transcriptional activity in panc-1 CSCs.</p><p>Conclusion</p><p>Gemcitabine treatment induced ER stress and promoted mutant p53-R273H stabilization via transcriptionally activated uPA which may contribute to chemoresistance to gemcitabine. Notably, upregulation of uPA by gemcitabine treatment may lead to the failure of CP-31398; thus, a novel strategy for modulating mutant p53 function needs to be developed.</p></div

Enrichment and characterization of human pancreatic CSCs from human pancreatic cancer cell line Panc-1.

<p>(A) Sphere formation of Panc-1 CSCs at day 7, 14 and 21. (B) Self-renewal capacity of Panc-1 CSCs were tested using serial replating assay. (C) Panc-1 CSCs were analyzed by folw cytometry using antibody against CD24, CD44, and CD133. (D) Expression of stem cell markers. The expression of CD24, CD44, CD133, Oct4, and Nanog was measured by RT-qPCR. Data represent mean±SD (left panel). protein levels were assessed by semi-quantitative Western blot (righ panel). (E) Gemcitabine sensitivity of Panc-1 cells or Panc-1 CSCs were assessed. *P<0.05; **P<0.01.</p

Gemcitabine treatment stimulated ER stress and activated transcriptional activity of ATF6α.

<p>(A) The expressing levels of pIRE1, IRE1, p-PERK, and PERK were measured by semi-quantitative Western blot with or without Gemcitabine treatment. (B) The respective band densitometry analyses were performed using the Image J software program. The values represent the mean±SD of three independent experiments. (C) The ratio of spliced XBP1 (XBP1/S) to XBP1 mRNA was calculated using the comparative Ct methods. (D) Protein of XBP1/S was detected by semi-quantitative Western blot. (E) The spliced form of ATF6α (p50ATF6) and unspliced form of ATF6α (p90ATF6) were detected. (F) Transcriptional activity of p50ATF6 was measured with the presence of PERK inhibitor (PERKi) or IRE1 inhibitor (IRE1i). *P<0.05.</p

uPA induced by Gemcitabine binds specifically to P53-R273H and partially promoted mitochondrial-dependent apoptosis.

<p>(A) RNA-IP assay was performed to confirm the binding of uPA mRNA 3’UTR to P53-R273H in Panc-1 CSCs. (B) EMSA assay was employed for confirming the direct and specific interaction between P53-R273H and fragment of uPA mRNA 3’UTR. (C) The mRNA (left panel) and protein (right panel) levels of uPA after p53 knockdown were measured. (D) The mRNA (left panel) and protein (right panel) levels of P53-R273H after uPA knockdown were measured. (E) The apoptotic condition was detected by flow cytometry using Annexin V-FITC/PI staining, after uPA or P53 knockdown. Caspase 3/7 activity (F) and cleaved PARP-1 (G) were analyzed for determine whether apoptosis induced is dependent on mitochondria. *P<0.05.</p