Hsa-miR-376c-3p targets Cyclin D1 and induces G1-cell cycle arrest in neuroblastoma cells

Abstract

High-risk neuroblastoma is the most aggressive form of cancer in children. The estimated survival of children with high-risk neuroblastoma is 40-50% compared with low and intermediate risk neuroblastoma, which is >98 and 90-95%, respectively. In addition, patients with high-risk neuroblastoma often experience relapse following intensive treatments with standard chemotherapeutic drugs. Therefore alternative strategies are required to address this problem. MicroRNAs (miRNAs/miRs) are small, endogenously expressed non-coding RNAs, which when deregulated have been demonstrated to serve significant roles in the tumorigenesis of a number of different types of cancer. Results from a previous deep sequencing study identified 22 downregulated miRNAs from the 14q32 miRNA cluster differentially expressed in neuroblastoma cell lines isolated from 6 patients at diagnosis and at relapse following intensive treatments. miR-376c-3p is one of the 22 miRNAs that was downregulated in the majority of the cell lines isolated from patients post treatment. The present study employed reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to quantify the basic expression of miR-376c-3p in 6 neuroblastoma cell lines. The functional role of miR-376c-3p in the neuroblastoma cell lines was evaluated by alamar blue-cell viability and propidium iodide-flow cytometric assays. In addition, luciferase reporter assays, RT-qPCR and western blotting were performed to identify and quantify the targets of miR-376c-3p in neuroblastoma cell lines. Ectopic expression of miR-376c-3p led to significant inhibition of cell viability and G1-cell cycle arrest in multiple neuroblastoma cell lines by reducing the expression of cyclin D1, an oncogene critical for neuroblastoma pathogenesis. The results of the present study provide novel insights into the functional role of miR-376c-3p and suggest new approaches for the treatment of neuroblastoma.

Keywords: 14q32 microRNA cluster; Cyclin D1; deep sequencing; high-risk neuroblastoma; microRNA; microRNA-376c-3p.

Figure 1.

(A) Schematic representation of the miRNA-cluster at 14q32 chromosomal region. Multiple miRNAs located at 14q32 chromosomal region are downregulated (bold type) and associated with poor prognosis factors in neuroblastoma. The miRNA of interest, miR-376c-3p is shown in bold and a rectangular box. (B) Box-plots of miR-376c expression in tumors from neuroblastoma patients at different stages. The boxes represent the 25–75% quartiles. The horizontal line in the boxes represents the median level. Whiskers represent the non-outlier range. Open circles represent the outliers. P=0.011, comparing the median expression level of miR376c-3p across all pathological stages. DLK1, Delta Like Non-canonical Notch Ligand 1; MEG, maternally expressed; snoRNA, small nucleolar RNAs; DIO3, iodothyronine deiodinase 3; miR/miRNA, microRNA.

Figure 2.

miR-376c-3p reduces cell viability in neuroblastoma cell lines. (A) RT-qPCR analysis was performed to quantitate the basic expression of miR-376c-3p in 6-neuroblastoma cell lines. miR-4286 served as an endogenous control for miRNAs. Error bars indicate mean ± SD of two independent experiments, each repeated in triplicate. (B) RT-qPCR analysis for confirmation of miR-376c-3p overexpression in 6-neuroblastoma cell lines transfected with NC or miR-376c-3p mimics. Expression of miR-376c-3p in SHSY-5Y cell line was set to 1 and miR-4286 served as an endogenous control for miRNAs. Error bars indicate mean ± SD. of two independent experiments, each repeated in triplicates. (C) Cell growth analysis of neuroblastoma cells transfected with NC or miR-376c-3p mimics. BE(2)-C, Kelly and SHSY-5Y were more sensitive compared with SKNAS, CHLA-15 and CHLA-20. Error bars indicate mean ± SD of three independent experiments repeated in triplicate. The results were statistically significant at 48, 72 and 96 h for all cell lines. *P<0.05. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; NC, negative control; SD, standard deviation; miR, microRNA.

Figure 3.

miR-376c-3p induces G1-cell cycle arrest in neuroblastoma cells. BE(2)-C, Kelly and SHSY-5Y cells were transfected with NC or miR-376c-3p mimics and the cell cycle analysis was evaluated by a propidium iodide-flow cytometric assay. The percentage of G1-arrested cells increased after miR-376c-3p overexpression. Error bars indicate mean ± standard deviation of three independent experiments repeated in triplicates. NC, negative control; miR, microRNA *P<0.05, **P<0.01 and ***P<0.001 vs. NC.

Figure 4.

CCND1 is a direct target of miR-376c-3p in neuroblastoma. (A) The CCND1 3′UTR contain one predicted miR-376c-3p binding site (nucleotides 1,792 to 1,798). In the figure, the alignment of the seed region of miR-376c-3p with CCND1 and the site of target mutagenesis are shown in bold and italics. (B) pMIR-Report-CCND1 luciferase constructs containing a full-length wt or mutated CCND1 3′UTR and miR-376c-3p or NC mimics, were co-transfected into BE(2)-C and SHSY-5Y cells. Luciferase activity of wt construct was significantly reduced compared with mutated constructs. Relative repression of firefly luciferase activity is normalized with renilla luciferase activity. Error bars indicate mean ± standard devaition of three independent experiments repeated in triplicates. *P<0.05, **P<0.01 vs. the adjacent NC. miR, microRNA; NC, negative control; CCND1, cyclin D1; ORF, open reading frame; UTR, untranslated region; wt, wild type; MUT, mutated.

Figure 5.

miR-376c-3p reduces mRNA and protein levels of CCND1 in neuroblastoma cells. (A) miR-376c-3p overexpression significantly decreases CCND1 mRNA levels in BE(2)-C, Kelly and SHSY-5Y. These cells were transfected with NC or miR-376c-3p mimics for 48 h and CCND1 levels were assessed by reverse transcription-quantitative polymerase chain reaction. (B) miR-376c-3p overexpression significantly decreases endogenous levels of Cyclin D1 proteins in BE(2)-C, Kelly and SHSY-5Y. These cells were transfected with NC or miR-376c-3p mimics for 96 h and Cyclin D1 expression assessed by western blot analysis. The β-actin antibody used as a loading control. (C) Quantitative analysis of Cyclin D1 protein expression on the western blots (n=3). Error bars indicate mean ± standard deviation of three independent experiments. *P<0.05 and **P<0.01 vs. the adjacent NC. miR, microRNA; NC, negative control; CCND1, cyclin D1.

Figure 6.

CCND1 overexpression partially rescues neuroblastoma cells from miR-376c-3p induced G1 cell cycle arrest. SHSY-5Y cells were co-transfected with NC or miR-376c-3p mimics and an empty or cyclin D1 overexpression plasmid. 96 h after the transfection, cells were ethanol fixed and cell cycle analysis was evaluated by the PI-flow cytometric assay. The results of the cell cycle analysis are depicted as percentage of cells in particular phases of cell cycle. Error bars indicate mean ± standard deviation of at least three repeats. miR, microRNA; NC, negative control; CCND1, cyclin D1.