miR-24 Inhibition Increases Menin Expression and Decreases Cholangiocarcinoma Proliferation

Abstract

Menin (MEN1) is a tumor-suppressor protein in neuroendocrine tissue. Therefore, we tested the novel hypothesis that menin regulates cholangiocarcinoma proliferation. Menin and miR-24 expression levels were measured in the following intrahepatic and extrahepatic cholangiocarcinoma (CCA) cell lines, Mz-ChA-1, TFK-1, SG231, CCLP, HuCCT-1, and HuH-28, as well as the nonmalignant human intrahepatic biliary line, H69. miR-24 miRNA and menin protein levels were manipulated in vitro in Mz-ChA-1 cell lines. Markers of proliferation and angiogenesis (Ki-67, vascular endothelial growth factors A/C, vascular endothelial growth factor receptors 2/3, angiopoietin 1/2, and angiopoietin receptors 1/2) were evaluated. Mz-ChA-1 cells were injected into the flanks of nude mice and treated with miR-24 inhibitor or inhibitor scramble. Menin expression was decreased in advanced CCA specimens, whereas miR-24 expression was increased in CCA. Menin overexpression decreased proliferation, angiogenesis, migration, and invasion. Inhibition of miR-24 increased menin protein expression while decreasing proliferation, angiogenesis, migration, and invasion. miR-24 was shown to negatively regulate menin expression by luciferase assay. Tumor burden and expression of proliferative and angiogenic markers was decreased in the miR-24 inhibited tumor group compared to controls. Interestingly, treated tumors were more fibrotic than the control group. miR-24-dependent expression of menin may be important in the regulation of nonmalignant and CCA proliferation and may be an additional therapeutic tool for managing CCA progression.

Figure 1

Menin is down-regulated in CCA. A and B: By real-time PCR and immunoblots, menin expression is decreased in CCA cell lines compared to H69. Significance is shown versus H69 cells. C: Flow cytometry analysis demonstrated a decrease in menin protein expression in Mz-ChA-1 cells compared to H69 cells. D: By real-time PCR, menin expression decreased in advanced-stage human CCA tissue biopsy specimens compared with normal control. Data are expressed as means ± SEM performed in triplicate (A–D). n = 3 independent samples (A and B). ^∗P < 0.05 versus H69 or human control. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 2

Increased menin expression decreases proliferation. Mz-ChA-1 cells overexpressing menin with pCMV6-MEN1 vector exhibit a decrease in Ki-67 proliferative marker expression. A–C: Increased menin expression in pCMV6-MEN1 Mz-ChA-1 cells by real-time PCR (A) and flow cytometry (B) decreased Ki-67 proliferative marker expression by real-time PCR (C). D: Decreased cell migration as measured by wound healing assay. E: Decreased cell invasion as measured by Boyden chamber assay in pCMV6-MEN1 Mz-ChA-1 cells. Data are expressed as means ± SEM performed in triplicate (A–E). ^∗P < 0.05 versus Mz-ChA-1 control cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 3

Menin expression negatively regulates angiogenesis. A: By real-time PCR, Mz-ChA-1 MEN1 knockout cells increased expression of angiogenic factors compared to Mz-ChA-1 control cells. B: By real-time PCR, pCMV6-MEN1 Mz-ChA-1 cells decreased expression of angiogenic factors compared to Mz-ChA-1 control cells. Data are expressed as means ± SEM performed in triplicate (A and B). ^∗P < 0.05 versus Mz-ChA-1 control cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 4

miR-24 negatively regulates menin. A: Real-time PCR evaluation of miR-24 expression in CCA and H69 cell lines demonstrates increased levels in CCA lines compared to H69 cells. B: Luciferase luminescence shows decreased menin expression with miR-24 mimic treatment. C: Left panel: miRNA-sequence data demonstrate increased expression of miR-24 in human CCA tumors compared with matched normal tissue. Right panel: Statistical significance of increased miR-24 expression is validated with an unpaired t-test. Data are expressed as means ± SEM (A–C). n = 3 (A and B); n = 9 (C). ^∗P < 0.05 versus normal matched human control.

Figure 5

miR-24 drives proliferation. A: Real-time PCR confirmed knockdown of miR-24 in Mz-ChA-1 cells by hairpin inhibitor. miR-24 knockdown increased menin expression via fluorescence-activated cell sorting (B) and decreased expression of angiogenic factors via real-time PCR (C). Data are expressed as means ± SEM performed in triplicate unless otherwise stated (A–C). ^∗P < 0.05 versus Mz-ChA-1 control cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 6

In vivo miR-24 inhibition decreases growth. A: Ten weeks after Mz-ChA-1 implantation into the flanks of nude mice, there was a significant reduction in tumor size in athymic mice treated with the miR-24 hairpin inhibitor compared to scramble mismatch-treated mice. Significance is shown versus mice treated with scramble mismatch. B: Real-time PCR performed on total RNA isolated from both tumor groups revealed decreased expression of proliferative and angiogenic factors. Significance is shown versus tumor samples from mismatch-treated mice. Data are expressed as means ± SEM of tumor size evaluations. n = 4 mice per each group (A); n = 3 experiments (B). ^∗P < 0.05 versus scramble mismatch control. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 7

In vivo miR-24 inhibition decreases growth. A and B: Immunohistochemical images of formalin-fixed, paraffin-embedded sections confirm expected histological depictions of Mz-ChA-1 CCA tumor via hematoxylin and eosin (H&E) and CK-19 staining. C–E: miR-24 inhibition decreased nuclear Ki-67 staining from 1 ± 0.15 inhibitor scramble to 0.4 ± 0.05 miR-24 inhibition (P = 0.001; C), decreased CD31 staining from 1 ± 0.29 inhibitor scramble to 0.1 ± 0.05 miR-24 inhibition (P = 0.02; D), and increased Sirius Red staining from 1 ± 0.47 inhibitor scramble to 10.7 ± 1.68 miR-24 inhibition (P = 0.003; E). Data are expressed as means ± SEM. n = 3 independent samples (A–E). P < 0.05 versus control inhibitor scramble. Original magnification: ×20 (A, B, and D); ×40 (C and E).