The miR-24-Bim pathway promotes tumor growth and angiogenesis in pancreatic carcinoma

Abstract

miRNAs are a group of small RNAs that have been reported to play a key role at each stage of tumorigenesis and are believed to have future practical value. We now demonstrate that Bim, which stimulates cell apoptosis, is obviously down-regulated in pancreatic cancer (PaC) tissues and cell lines. And Bim-related miR-24 is significantly up-regulated in PaC. The repressed expression of Bim is proved to be a result of miR-24, thus promoting cell growth of both cancer and vascular cells, and accelerating vascular ring formation. By using mouse tumor model, we clearly showed that miR-24 promotes tumor growth and angiogenesis by suppressing Bim expression in vivo. Therefore, a new pathway comprising miR-24 and Bim can be used in the exploration of drug-target therapy of PaC.

Keywords: Bim; angiogenesis; miR-24; pancreatic cancer; tumorigenesis.

Figure 1. The expression patterns of Bim and miR-24 in PaC tissues

A. Western blotting analysis of Bim protein levels in human PaC tissues (n = 6). B. Quantitative analysis of A. C. Relative levels of Bim mRNA in human PaC tissues (n = 6). D. Immunohistochemistry of the paraffin-embedded human pancreatic tumor tissues. E. The two predicted binding sites of miR-24 in the 3′UTR of Bim. F. Schematic description of the base-pairing interaction between miR-24 and Bim mRNA. G. Quantitative RT-PCR analysis of miR-24 levels in PaC tissues (n = 6). ** indicates p < 0.01.

Figure 2. miR-24 suppresses Bim expression in PANC1 cells

A. Relative levels of miR-24 in PANC1 cells transfected with mimics or inhibitors (n = 3). B. and C. Direct recognition of Bim 3′UTR by miR-24. PANC1 cells were co-transfected with firefly luciferase reporters containing either WT or mutant Bim 3′UTR with miR-24 mimics, inhibitors and the corresponding normal control. Cells were assayed using a luciferase assay kit at 24 h after transfection. The target 1 B.and target 2 C. of miR-24 were detected respectively (n = 3). D. and E. miR-24 suppresses Bim expression in PANC1 cells. Bim mRNA levels were assessed by quantitative RT-PCR D. and protein levels were analyzed by western blotting E. (n = 3). *** indicates p < 0.001; ** indicates p < 0.01; * indicates p < 0.05.

Figure 3. miR-24 promotes cell growth while inhibits apoptosis of PANC1 cells

A. and B. miR-24 increases cell ratio in S phase. PANC1 cells were transfected with miR-24 mimics and cell cycle were evaluated by cell flow assay B. cells treated with water were used as normal control A. C. Quantified analysis of A and B (n = 5). D. miR-24 promotes cell proliferation of PANC1 cells (n = 5). E. miR-24 suppresses cell apoptosis (n = 5). ** indicates p < 0.01; * indicates p < 0.05.

Figure 4. Knock-down of Bim in PANC1 cells

Bim siRNA was transfected into PANC1 cells. A. and B. Bim protein A. and mRNA B. levels in PANC1 treated with Bim siRNA. C. and D. Effects of Bim siRNA on cell proliferation C. and apoptosis D. in PANC1 cells. n = 5; ** indicates p < 0.01.

Figure 5. The miR-24-Bim pathway regulates angiogenesis

A. and C. Bim protein A. and mRNA levels C. in HUVEC cells (n = 5). B. Relative levels of miR-24 in HUVEC cells treated with miR-24 mimics or inhibitors (n = 5). D. Representative images of HUVEC cells on Matrigel. E. Quantitative analysis of the experiments in panel D (n = 5). F. and G. Cell proliferation by performing an Edu incorporation assay (n = 5). H. and I. Cell apoptosis measured by flow cytometry (n = 5). ** indicates p < 0.01.

Figure 6. Effects of miR-24-Bim pathway on tumor growth in vivo

A. Morphology of the tumors from tumor-implanted nude mice (n = 6). B. Weight of tumors excised from mice implanted with control PANC1 cells, miR-24-overexpressing PANC1 cells and Bim-overexpressing PANC1 cells. (n = 6) C. Relative levels of miR-24 in implanted tumor tissues (n = 6). D. and E. The Bim mRNA and protein levels in implanted tumor tissues (n = 6). F. Immunohistochemistry of the paraffin-embedded pancreatic tumor tissues shown in A (n = 6). ** indicates p < 0.01.