MiR-543/Numb promotes proliferation, metastasis, and stem-like cell traits of prostate cancer cells

Abstract

MiR-543 and Numb are associated with various malignancies, including prostate cancer (PCa). However, whether miR-543 regulates Numb in PCa development remains unclear. In this study, we identified Numb as a direct target of miR-543. The role of miR-543 was examined both in vitro and in vivo. The in vivo effects of miR-543 were investigated using tumor transplantation experiments and a lung metastasis model. The in vitro effects of miR-543 on proliferation, migration, invasion, and cancer stem-like cell (CSC)-associated properties were also examined. The binding sites of Numb were predicted using bioinformatics tools and confirmed by luciferase and rescue assays. QRT-PCR and western blot analyses were used to detect target expression levels. Expression levels of both miR-543 and Numb were manipulated in CD44+ and CD44-PCa cells followed by a series of functional assays. The results demonstrated that miR-543 promoted PCa growth and metastasis both in vivo and in vitro. Luciferase reporter assays, qRT-PCR, and western blot analyses revealed Numb as a direct target of miR-543. The function of miR-543 was abolished by Numb, as shown in rescue experiments. Moreover, miR-543 was verified to promote CSC properties, whereas Numb elicited the opposite effects. MiR-543 also influenced the expression of several stem-like factors, including Dll4, NF-κB, c-myc, and Oct-4, and the Numb/p53 signaling pathway. Taken together, these results demonstrate that miR-543 plays an oncogenic role by negatively controlling Numb, revealing the existence of an miR-543/Numb/p53 regulatory pathway in PCa tumorigenesis and development.

Keywords: CSC; Numb; miR-543; proliferation; prostate cancer.

Figure 1

Overexpression of miR-543 promotes the growth of tumor xenografts in vivo. A. The images show tumors harvested from BALB/C nude mice that were subcutaneously injected with PC3 or DU145 cells transfected with the miR-543 mimic, mimic NC, miR-543 inhibitor (anti-543), or inhibitor NC (anti-NC). The tumor image, incidence (tumors/injections), final weights of the harvested tumors (mean ± S.D.), and their corresponding P values are shown on the left. B. Representative images of the immunohistochemical staining for Ki-67 and caspase-3 in harvested tumor tissues are shown on the bottom (magnification, × 200). C. Percentage analysis of cells staining positive for Ki67 and caspase-3 is shown on the right. Results are expressed as the means ± S.D. from three independent replicates, *P < 0.05.

Figure 2

MiR-543 is significantly associated with pulmonary metastasis in a tail vein injection model. A. Images show lung tissues harvested 42 days after injecting PC3 cells transfected with the miR-543 mimic or mimic NC; arrows indicate visible metastatic lung nodules. B. The middle image shows the quantitative analysis of the number of metastatic lung nodules. C. HE staining of metastatic lung nodules from mice 42 days after injection with miR-543 mimic- and NC-transfected PC3 cells. Results are expressed as the means ± S.D. from three independent replicates, *P < 0.05.

Figure 3

MiR-543 overexpression promotes PCa cell invasion and migration in vitro. A. A Transwell invasion assay was performed to compare the invasive abilities of different groups in PC3 and DU145 cells transfected with the miR-543 mimic, mimic NC, miR-543 inhibitor (anti-543), or inhibitor NC (anti-NC). B. A Transwell migration assay was performed to compare the migration abilities of the different groups. C. The invasion of cells from the different groups were analyzed. D. The migration of cells from the different groups were analyzed. E. Western blotting was used to detect differences in E-cadherin and vimentin protein levels in PCa cells transfected with the miR-543 mimic, miR-543 mimic, or their corresponding NC. Results are expressed as the means ± S.D., *P < 0.05.

Figure 4

MiR-543 overexpression promotes clonal, clonogenic, and sphere-forming activities of PCa cells in vitro. A. PC3 and DU145 cells transfected with the miR-543 mimic or NC were plated onto a six-well plate for 48 hours, and clonal experiments were performed. B. The two cell lines were transfected with the miR-543 mimic, mimic NC, inhibitor (anti-543), or inhibitor NC (anti-NC) for 48 hours, and the 3D Matrigel clonogenic assay was performed. C. The sphere-formation assay was performed in transfected PC3 and DU145 cells to determine the self-renewal ability of the CSCs. All results are expressed as the means ± S.D. from three independent replicates. *P < 0.05.

Figure 5

Numb is a direct and functional target of miR-543 in PCa cells. (A) The schematic diagram shows the region where miR-543 is believed to bind the Numb 3’-UTR and the corresponding mutation of Numb in the luciferase reporter assay. (B) Dual luciferase assays showed that luciferase activity was inhibited by miR-543. Cells were co-transfected with miR-128 or NC, and reporter vectors containing empty (Ctrl), wild-type (Wt), or mutant (Mut) Numb for 48 hours. (C-F) PC3 cells were co-transfected with the miR-543 mimic and Numb cDNA lacking the 3’-UTR to restore the expression of Numb for 48 hours, and then the migration (C), invasion (D), and clonal (E) and sphere-formation abilities (F) of PC3 cells were measured. The results are represented as the means ± SEM of three independent assays, *P < 0.05, **P < 0.01.

Figure 6

MiR-543 regulates the expression of Numb and several stem-like regulators. A. A qPCR assay was performed to assess miR-543 levels in CD44+ DU145 and CD44+ PC3 cells compared to CD44- PCa cells. B. A qPCR assay was performed to assess Numb levels in CD44+ DU145 and CD44+ PC3 cells compared to CD44- PCa cells. C. The effects of miR-543 overexpression are shown on Numb expression and the expression of several stem cell regulators in DU145 and PC3 cells. D. Western blotting was performed to assess the effects of miR-543 overexpression on Numb, p53, and Notch1 levels. E. Numb protein was knocked down using Numb siRNA-1 and Numb siRNA-2. The results are represented as the means ± SEM of three independent assays, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

MiR-543 and Numb exert opposite effects in CD44+ PCSCs. A-C. Transwell invasion assays, 3D Matrigel clonogenic assays, and sphere-formation assays were performed in CD44+ PC3 cells transfected with the miR-543 inhibitor (anti-543) or inhibitor NC (anti-NC). D-F. Transwell invasion assays, 3D Matrigel clonogenic assays, and sphere-formation assays were respectively performed in CD44- PC3 cells transfected with the miR-543 mimic or mimic NC. G. Numb protein levels in CD44+ PC3 cells transfected with Numb vector containing full-length Numb. H-J. Transwell invasion assays, 3D Matrigel clonogenic assays, and sphere-formation assays were performed in pcDNA3.1-Numb- and pcDNA3.1-Ctrl-transfected CD44+ PC3 cells. K. Numb protein levels in CD44- PC3 cells transfected with Numb siRNA. L-N. Transwell invasion assays, 3D Matrigel clonogenic assays, and sphere-formation assays were performed in CD44- PC3 cells transfected with Numb siRNA or NC siRNA. The results are represented as the means ± SEM of three independent assays, *P < 0.05.