miR-124 suppresses proliferation and invasion of nasopharyngeal carcinoma cells through the Wnt/β-catenin signaling pathway by targeting Capn4

Abstract

Background: Recent studies have demonstrated that microRNA 124 (miR-124) acts as a tumor suppressor in nasopharyngeal carcinoma (NPC); however, the exact molecular mechanism by which miR-124 exerts tumor suppression has not been well elucidated.

Materials and methods: We performed quantitative real-time PCR (qRT-PCR) to measure the expression of metastasis associated lung adenocarcinoma transcript 1, miR-124, and calpain small subunit 1 (Capn4) mRNAs in NPC cell lines. We also performed western blot analysis to detect the levels of Capn4. Furthermore, we performed MTT assay and transwell invasion assay to determine the proliferation and invasion ability of two NPC cell lines, namely, HONE1 and CNE2 cells, respectively. The verification of targets of miR-124 was performed using prediction softwares and luciferase reporter analysis.

Results: According to our results, the expression of Capn4 was found to be elevated, whereas the expression of miR-124 was lowered in NPC cell lines compared with normal nasopharyngeal cells. When we preformed overexpression of miR-124, it suppressed the proliferation and invasion of NPC cells. Moreover, miR-124 suppressed the expression of Capn4 by targeting Capn4 in HONE1 and CNE2 cells. When we preformed overexpression of Capn4, it reversed the inhibitory effect of miR-124 on the proliferation and invasion of NPC cells. Furthermore, miR-124-Capn4 axis decreased the levels of β-catenin, cyclin D1, and c-Myc, the components of the Wnt/β-catenin signaling pathway.

Conclusion: The suppression of proliferation and invasion of NPC cells by miR-124 were achieved by the regulation of Wnt/β-catenin signaling pathway by targeting Capn4. The results of this study revealed a novel miR-124-Capn4 regulatory axis in NPC cell lines, providing a better understanding of the pathogenesis of NPC and a promising therapeutic target for patients with NPC.

Keywords: Calpain small subunit 1; NPC; miR-124.

Figure 1

The level of microRNA-124 (miR-124) was downregulated and Capn4 expression was upregulated in NPC cell lines (HONE1, CNE1, and CNE2) compared with human nasal epithelial cell line (HNEpC) or immortalized nasopharyngeal epithelial cell line (NP69). Notes: (A) Quantitative real-time PCR (qRT-PCR) analysis revealed decreased miR-124 expression in HONE1, CNE1, and CNE2 cells. (B) qRT-PCR analysis indicated that the expression level of Capn4 mRNA was elevated in HONE1, CNE1, and CNE2 cells. (C) Western blot analysis showed the increased protein level of Capn4 in HONE1, CNE1, and CNE2 cells. Data are shown as mean ± standard deviation (n=3). *P<0.05 NPC cells vs HNEpC or NP69.

Figure 2

MicroRNA-124 (miR-124) inhibited proliferation and invasion of nasopharyngeal carcinoma (NPC) cell lines HONE1 and CNE2. HONE1 and CNE2 cells were transfected with miR-control or miR-124. Notes: (A) MTT assay revealed that the cell viability decreased 24, 48, and 72 h after miR-124 transfection in HONE1 and CNE2 cells when compared with the miR-control or untreated cells (NC group). Data are shown as mean ± standard error of mean (n=3). *P<0.05 miR-124 treated cells vs miR-control treated cells or untreated cells. (B) Transwell invasion assay showed that the invasion of HONE1 and CNE2 cells was suppressed after transfection with miR-124. Data are shown as mean ± standard deviation (n=3). *P<0.05 miR-124 treated cells vs miR-control treated cells. Abbreviation: NC, untreated cells.

Figure 3

MicroRNA-124 (miR-124) directly suppressed Calpain small subunit 1 (Capn4) expression. Notes: (A) Putative miR-124 binding sequence of Capn4 was shown. (B) The relative luciferase activity was detected in HONE1 and CNE2 cells co-transfected with wild-type or mutant Capn4 3′UTR and miR-124 mimic or miR-control. *P<0.05 (C and D) Western blot analysis indicated that the level of Capn4 expression was decreased by miR-124 transfection and elevated by miR-124 inhibition in HONE1 and CNE2 cells. NC represents untreated cells. Data are shown as mean ± standard deviation (n=3). *P<0.05 miR-124 treated cells vs miR-control treated cells. Abbreviations: MUT, mutant; NC, untreated cells; WT, wild-type.

Figure 4

MicroRNA-124 (miR-124) suppressed proliferation and invasion of NPC cells through the inhibition of Capn4 expression. HONE1 and CNE2 cells were transfected with miR-124 or in combination with pcDNA-Capn4. Notes: (A) MTT assay confirmed that restored Capn4 expression reversed the inhibitory effect of miR-124 on the cell viability of HONE1 and CNE2 cells at 24, 48, and 72 h after transfection. Data are shown as mean ± standard error of mean (n=3) (B and C) Transwell invasion assay suggested that transfection of pcDNA-Capn4 abolished miR-124-mediated inhibition of cell invasion in HONE1 and CNE2 cells. Data are shown as mean ± standard deviation (n=3). *P<0.05. Abbreviation: NPC, nasopharyngeal carcinoma.

Figure 5

The Wnt/β-catenin signaling pathway inhibitor, XAV939, suppressed proliferation and invasion of nasopharyngeal carcinoma (NPC) cells. HONE1 and CNE2 cells were treated with different doses of XAV939 (0, 10, and 20 μM). Notes: (A) MTT assay displayed that the cell viability of HONE1 and CNE2 cells was inhibited by XAV939 in a dose-dependent manner at 24, 48, and 72 h after XAV939 treatment. Data are shown as mean ± standard error of mean (n=3) *P<0.05. (B) Transwell invasion assay indicated that the invasion ability of HONE1 and CNE2 cells was inhibited by XAV939 in a dose-dependent manner after treatment with XAV939 for 48 h. Data are shown as mean ± standard deviation (n=3). *P<0.05 XAV939 treated cells vs control media treated cells.

Figure 6

Overexpression of calpain small subunit 1 (Capn4) reversed the inhibitory effect of miR-124 on the Wnt/β-catenin signaling pathway. HONE1 and CNE2 cells were transfected with miR-124 or co-transfected with miR-124 and pcDNA-Capn4 (A and B). Western blot analysis revealed that Capn4 overexpression reversed the decreased β-catenin, cyclin D1, and c-Myc protein levels caused by the overexpression of miR-124 in HONE1 and CNE2 cells. Data are shown as mean ± standard deviation (n=3). *P<0.05 miR-124 or pcDNA -Capn4 treated cells vs miR-control or vector treated cells.