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. 2009 Aug 28;4(8):e6816.
doi: 10.1371/journal.pone.0006816.

MicroRNA miR-34 inhibits human pancreatic cancer tumor-initiating cells

Qing Ji  1 , Xinbao Hao, Min Zhang, Wenhua Tang, Meng Yang, Ling Li, Debing Xiang, Jeffrey T Desano, Guido T Bommer, Daiming Fan, Eric R Fearon, Theodore S Lawrence, Liang Xu
Affiliations

MicroRNA miR-34 inhibits human pancreatic cancer tumor-initiating cells

Qing Ji et al. PLoS One. .

Abstract

Background: MicroRNAs (miRNAs) have been implicated in cancer initiation and progression via their ability to affect expression of genes and proteins that regulate cell proliferation and/or cell death. Transcription of the three miRNA miR-34 family members was recently found to be directly regulated by p53. Among the target proteins regulated by miR-34 are Notch pathway proteins and Bcl-2, suggesting the possibility of a role for miR-34 in the maintenance and survival of cancer stem cells.

Methodology/principal findings: We examined the roles of miR-34 in p53-mutant human pancreatic cancer cell lines MiaPaCa2 and BxPC3, and the potential link to pancreatic cancer stem cells. Restoration of miR-34 expression in the pancreatic cancer cells by either transfection of miR-34 mimics or infection with lentiviral miR-34-MIF downregulated Bcl-2 and Notch1/2. miR-34 restoration significantly inhibited clonogenic cell growth and invasion, induced apoptosis and G1 and G2/M arrest in cell cycle, and sensitized the cells to chemotherapy and radiation. We identified that CD44+/CD133+ MiaPaCa2 cells are enriched with tumorsphere-forming and tumor-initiating cells or cancer stem/progenitor cells with high levels of Notch/Bcl-2 and loss of miR-34. More significantly, miR-34 restoration led to an 87% reduction of the tumor-initiating cell population, accompanied by significant inhibition of tumorsphere growth in vitro and tumor formation in vivo.

Conclusions/significance: Our results demonstrate that miR-34 may restore, at least in part, the tumor suppressing function of the p53 in p53-deficient human pancreatic cancer cells. Our data support the view that miR-34 may be involved in pancreatic cancer stem cell self-renewal, potentially via the direct modulation of downstream targets Bcl-2 and Notch, implying that miR-34 may play an important role in pancreatic cancer stem cell self-renewal and/or cell fate determination. Restoration of miR-34 may hold significant promise as a novel molecular therapy for human pancreatic cancer with loss of p53-miR34, potentially via inhibiting pancreatic cancer stem cells.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Expression of Bcl-2 family of proteins and miR-34s in human pancreatic cancer cell lines as well as normal human fibroblast cells WI-38.
A, Western blot analysis. B, qRT-PCR analysis of relative expression levels of miR-34s. C, qRT-PCR analysis of the expression levels of miR-34 target genes in human pancreatic cancer cell lines as well as normal human fibroblast WI-38 cells. The cells were lyzed to extract total RNA for qRT-PCR, data were normalized to that of Actin and the relative levels are shown (Actin = 1000). Note p21 is a target gene of p53.
Figure 2
Figure 2. Restoration of miR-34 down-regulates target genes' expression.
A, miR-34 restoration down-regulates target proteins Bcl-2, Notch1 and Notch2, no effects on Mcl-1. MiaPaCa2 cells were transfected with miR-34 mimics or non-specific control miRNA mimic (NC mimic) (100 pmol per well in 6-well plates by Lipofectamine 2000) for 48 hours, then collected for Western blot analysis. B, Quantitative real-time PCR analysis of the potential target genes' mRNA levels after miR-34 mimic transfection in MiaPaCa2 cells. **P<0.01, ***P<0.001, Student's t-test, n = 2. C, Bcl-2 3′UTR Luciferase Reporter Assay shows that the transfected miR-34 mimics are functional. MiaPaCa2 cells were co-transfected with the Bcl-2 3′UTR Luciferase Reporter or its mutant, b-gal vector, together with either miR-34 mimics or NC mimic. Luciferase assay was performed 24 hrs after transfection using Bright-Glo Luciferase Assay System. Luciferase activity was normalized relative to b-gal activity. Error bar indicates s.e.m.
Figure 3
Figure 3. Restoration of miR-34 inhibits the clonogenic growth of MiaPaCa2 cells, whereas inhibition of miR-34 promotes cell growth.
MiaPaCa2 cells were transfected with miR-34 mimics or inhibitors, 24 hr later the cells were seeded in 6-well plates (200 cells/well, in triplicates). After 12–14 days incubation, the plates were gently washed with PBS and stained with 0.1% crystal violet. A, representative pictures of the colonies. B, Colonies with over 50 cells were counted. C, Restoration of miR-34 leads to caspase-3 activation. Caspase-3 activation assay was carried out as described in in Materials and Methods . Fold increase of fluorescence signal was calculated by dividing the normalized signal in each treated sample with that in the untreated control. **P<0.01, ***P<0.001, Student's t-test, n = 3. D, Cell cycle distribution of MiaPaCa2 cells transfected with miR-34 mimics. Cell cycle analysis was performed 1 day after transfection. Cells were stained with propidium iodide after ethanol fixation and analyzed by flow cytometry.
Figure 4
Figure 4. Restoration of miR-34 sensitizes MiaPaCa2 cells to chemotherapy and radiation.
A, miR-34 restoration sensitizes the cells to chemotherapeutic agents. The MTT-based cytotoxicity assay was carried out using the Zeocin-resistant stable MiaPaCa2-miR-34a-MIF and MiaPaCa2-MIF cells. B, miR-34 restoration increases caspase-3 activation induced by gemcitabine or X-ray radiation in MiaPaCa2 cells. Relative caspase-3 activation was calculated by normalizing the fluorescence signal in each treated sample with that of the NC mimic or MIF control as 100. *P<0.05, ***P<0.001, Student's t-test, n = 3. C, miR-34 restoration increases radiation-induced apoptosis in MiaPaCa-2 cells. Cells were transfected with miR-34a mimic or NC mimic. 24 hr later, the cells were subjected to X-ray radiation. The cells were collected after another 48 hr, stained with propidium iodide after ethanol fixation, and analyzed by flow cytometry for the % of cells in sub-G1 phase. *P<0.05, Student's t-test, n = 2. D, miR-34 restoration radiosensitized MiaPaCa-2 cells. The clonogenic assay was carried out as described in Materials and Methods Data are shown as mean +/− SD (n = 3).
Figure 5
Figure 5. MiaPaCa2 CD44+/CD133+ cells are tumorsphere-forming cells that have high Bcl-2 and loss of miR-34.
A, CD44 and CD133 staining of MiaPaCa2 cells. MiaPaCa2 cells were stained by anti-CD44-APC and anti-CD133-PE and sorted by FACS. Around 1–2% cells are CD44+/CD133+ double-positive (Q2). B–C, Tumorsphere culture of the sorted cells. The sorted cells were plated for tumorsphere culture as described in Materials and Methods . 7–10 days later, tumorspheres were counted (B). The insert shows a representative tumorsphere from CD44+/CD133+ MiaPaCa2 cells. C. Quantification of cell numbers per tumorsphere. Tumorspheres were collected with 40 um filter and dissociated with trypsin to make a single cell suspension. Cells were counted with trypan blue exclusion and data are presented as number of cells per tumorsphere. CD44+/CD133+ cells are tumorsphere-forming cells whereas CD44−/CD133− cells did not grow tumorspheres. *P<0.05, **P<0.01, ***P<0.001, Student's t-test, n = 3. D, qRT-PCR analysis of the expression levels of miR-34 and target genes in the sorted MiaPaCa2 cells. The sorted cells were lyzed to extract total RNA for qRT-PCR. The miR-34 expression data were normalized to that of Actin and the relative levels are shown (set unsorted cells = 1). For the target genes expression, data were normalized to that of Actin (set Actin = 1000). CD44+/CD133+ (Q2) cells have high levels of Bcl-2 and Notch1 but loss of miR-34a/b/c as compared with CD44−/CD133− (Q3) cells. *P<0.05, **P<0.01, ***P<0.001, Student's t-test, n = 2.
Figure 6
Figure 6. Restoration of miR-34 by MIF lentiviral system decreases the CD44+/CD133+ MiaPaCa2 cells and inhibits tumorspheres from the sorted CD44+/CD133+ cells.
A–B, CD44 and CD133 FACS analysis of the MiaPaCa2-miR-34a-MIF and MiaPaCa2-MIF cells. miR-34a restoration significantly reduced the CD44+/CD133+ cells. Values are mean±s.e.m, n = 2. C, miR-34a restoration inhibits tumorspheres from the sorted CD44+/CD133+ cells. The cell sorting and tumorsphere culture were as described in Materials and Methods . 7–10 days later, tumorspheres were counted and the cell numbers per tumorsphere were quantified. Tumorspheres were collected with 40 um filter (BD) and dissociated with trypsin to make a single cell suspension. Cells were counted with trypan blue exclusion and data are presented as number of cells per tumorsphere. *P<0.05, Student's t-test, n = 3. D, qRT-PCR analysis of Bcl-2 mRNA levels in the sorted cells with or without miR-34a restoration. Data are shown as relative mRNA levels normalized to that of Actin = 1000 arbitrary units. Values are mean±s.d, n = 2. miR-34a restoration led to almost 23-fold reduction of Bcl-2 mRNA in CD44+/CD133+ cells, compared with 43% reduction in total population. **P<0.01, ***P<0.001, one-way ANOVA and Student's t-test, n = 2.
Figure 7
Figure 7. Restoration of miR34a inhibits the clonogenic growth and tumorspheres of BxPC-3 cells.
BxPC-3 cells were infected with lentiviral miR-34a expression system (miR-34a) or vector control (MIF), and the infected cells were sorted for GFP positive cells by FACS. The sorted cells were plated for either colony formation (A) or tumorsphere culture (B) as described in Materials and Methods . **P<0.01, ***P<0.001, Student's t-test, n = 3.
Figure 8
Figure 8. miR-34 restoration inhibits the MiaPaCa2 tumor initiation in nude mice.
MiaPaCa2 cells were transfected with miR-34a mimic or NC mimic for 24 hours. Cells were collected and inoculated into female athymic nude mice subcutaneously (s.c.) on both sides of flank, 1×106 cells/0.2 ml. The tumor sizes were measured using a caliper (A). Tumor volume was calculated using the formula: (length×width2)/2. On Day 38, all tumors were collected to measure the tumor weights (B). C, Picture of the tumors. ***P<0.0001, two-way ANOVA, n = 10.
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