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. 2016 Jul 19;7(29):45547-45561.
doi: 10.18632/oncotarget.9999.

MicroRNA-1291 Targets the FOXA2-AGR2 Pathway to Suppress Pancreatic Cancer Cell Proliferation and Tumorigenesis

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Free PMC article

MicroRNA-1291 Targets the FOXA2-AGR2 Pathway to Suppress Pancreatic Cancer Cell Proliferation and Tumorigenesis

Mei-Juan Tu et al. Oncotarget. .
Free PMC article

Abstract

Pancreatic cancer is the fourth leading cause of cancer death in the United States. Better understanding of pancreatic cancer biology may help identify new oncotargets towards more effective therapies. This study investigated the mechanistic actions of microRNA-1291 (miR-1291) in the suppression of pancreatic tumorigenesis. Our data showed that miR-1291 was downregulated in a set of clinical pancreatic carcinoma specimens and human pancreatic cancer cell lines. Restoration of miR-1291 expression inhibited pancreatic cancer cell proliferation, which was associated with cell cycle arrest and enhanced apoptosis. Furthermore, miR-1291 sharply suppressed the tumorigenicity of PANC-1 cells in mouse models. A proteomic profiling study revealed 32 proteins altered over 2-fold in miR-1291-expressing PANC-1 cells that could be assembled into multiple critical pathways for cancer. Among them anterior gradient 2 (AGR2) was reduced to the greatest degree. Through computational and experimental studies we further identified that forkhead box protein A2 (FOXA2), a transcription factor governing AGR2 expression, was a direct target of miR-1291. These results connect miR-1291 to the FOXA2-AGR2 regulatory pathway in the suppression of pancreatic cancer cell proliferation and tumorigenesis, providing new insight into the development of miRNA-based therapy to combat pancreatic cancer.

Keywords: AGR2; FOXA2; miR-1291; microRNA; pancreatic cancer.

Conflict of interest statement

The authors have no conflicts of interests to declare.

Figures

Figure 1
Figure 1. miR-1291 expression levels are lower in human pancreatic cancer cell lines and patient PDAC tissues
(A) The expression levels of miR-1291 were remarkably lower in human pancreatic cancer cell lines than other cell lines. ***P < 0.001, compared to HepG2 cells. Values are mean ± SD (N = 3). (B) The average expression level of miR-1291 was about 60% lower in PDAC tissues than unpaired non-tumor tissues, and over 6-fold lower than paired peripheral non-tumor tissues. *P < 0.05, and **P < 0.01; values are mean ± SD.
Figure 2
Figure 2. Restoration of miR-1291 expression suppresses the proliferation of PANC-1 and AsPC-1 cells
(A, B) miR-1291 expression levels were about 9- and 12-fold higher in miR-1291 stably transfected PANC-1 and AsPc-1 cells, respectively, as compared to corresponding control cells transfected with empty vectors. (C, D) Cell proliferation capacity was significantly reduced in the miR-1291-expressing PANC-1 and AsPC-1 cells, as determined by MTT assays. Viability of control cells at the last time point was set as 100%. Values are mean ± SD (N =3). ***P < 0.001, *P < 0.05, compared to control cells.
Figure 3
Figure 3. Reintroduction of miR-1291 into PANC-1 cells induces a G2/M cell cycle arrest and an enhanced apoptosis
(A, B) Comparison of flow cytometry histograms of control and miR-1291-expressing PANC-1 cells stained with propidium iodide, and (C) the percentage of cells at various phases (G1/G0, S and G2/M). (D, E) Comparison of flow cytometry histograms of control and miR-1291-expressing cells stained with Annexin V/propidium iodide, and (F) the percentage of apoptotic cells. Values are mean ± SD (N = 3). ***P < 0.001,*P < 0.05, compared to corresponding controls.
Figure 4
Figure 4. miR-1291 suppresses the tumorigenicity of PANC-1 cells in xenograft tumor mouse models
(A) Representative picture of mouse inoculated with control and miR-1291-expressing PANC-1 cells. (B) Growth of xenograft tumors from miR-1291-expressing PANC-1 cells was significantly (P < 0.001, two-way ANOVA) slower than the control cells. **P < 0.01 and *P < 0.05, compared to the same time points. (C) Visual comparison of xenograft tumors excised from individual mice at week 8 after inoculation. (D) Xenograft tumors derived from miR-1291-expressing PANC-1 cells was significantly (***P < 0.001) lighter than the control cells. Values are mean ± SD (N = 12).
Figure 5
Figure 5. Difference in global protein expression profiles between miR-1291-expressing and control PANC-1 cells
(A–C) 2D-DIGE images of proteins in the control and miR-1291-expression PANC-1 cells labeled with green and red fluorescent dye, respectively, and the overlaid graph indicating the difference in the abundance of proteins. The protein downregulated in miR-1291-expressing PANC-1 cells to the greatest degree was identified as AGR2. (D, E) Immunocytochemistry analysis confirmed the sharp downregulation of AGR2 (brown staining) in miR-1291-expressing PANC-1 cells (400 ×).
Figure 6
Figure 6. The network of interactions among miR-1291-modualted proteins
Network of interactions was constructed using IPA software. The input was all miR-1291-altered proteins (in bold) in PANC-1 cells identified by 2D-DIGE, MALDI-TOF and MS/MS proteomic profiling study.
Figure 7
Figure 7. AGR2 is overexpressed in patient PDAC tissues, which is in contrast to the reduced expression of miR-1291 (Figure 1B)
(A, B) AGR2 mRNA levels were significantly higher in pancreatic tumor tissues than non-tumor tissues, as determined by selective qPCR analyses. (C) AGR2 protein (brown stains) levels were much higher in patient PDAC than adjunct normal pancreatic tissues, as demonstrated by immunohistochemistry study.
Figure 8
Figure 8. miR-1291 modulates AGR2 expression through targeting of FOXA2, a known transcriptional regulator of AGR2
(A, B) Western blot analysis showed that both FOXA2 and AGR2 protein levels were much lower in miR-1291-expressing PANC-1 (A) and AsPC-1 (B) cells, as compared to corresponding control cells. GAPDH was used as a loading control. (C) Computational analysis revealed two putative MRE sites for miR-1291 within the 3′UTR of FOXA2. Underlined is the seed sequence of miR-1291. (D, E) FOXA2 3′UTR luciferase reporter activities were decreased in PANC-1 cells (D) and increased in HepG2 cells (E) after the transfection with miR-1291 expression plasmid and antagomir, respectively. *P < 0.05, compared to the control group. Values are mean ± SD (N = 9).

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