The miR-181 family promotes cell cycle by targeting CTDSPL, a phosphatase-like tumor suppressor in uveal melanoma

Abstract

Background: MicroRNAs (miRNAs) have been shown to function in many different cellular processes, including proliferation, apoptosis, differentiation and development. miR-181a, -181b, -181c and -181d are miR-181 members of the family, which has been rarely studied, especially uveal melanoma.

Methods: The expression level of miR-181 family in human uveal melanoma cell lines was measured via real-time PCR (RT-PCR). The function of miR-181 on cell cycle was detected through Flow Cytometry assay. Microarray assay and Bioinformatics analysis were used to find the potential target of miR-181b, and dual-luciferase reporter assays further identified the target gene.

Results: MiR-181 family members were found to be highly homologous across different species and their upregulation significantly induces UM cell cycle progression. Of the family members, miR-181b was significantly overexpressed in UM tissues and most UM cells. Bioinformatics and dual luciferase reporter assay confirmed CTDSPL as a target of miR-181b. miR-181b over-expression inhibited CTDSPL expression, which in turn led to the phosphorylation of RB and an accumulation of the downstream cell cycle effector E2F1, promoting cell cycle progression in UM cells. Knockdown CTDSPL using siRNAs showing the same effect, including increase of E2F1 and the progression of cell cycle.

Conclusions: MiR-181 family members are key negative regulators of CTDSPL-mediated cell cycle progression. These results highlight that miR-181 family members, especially miR-181b, may be useful in the development of miRNA-based therapies and may serve as novel diagnostic and therapeutic candidate for UM.

Keywords: CTDSPL; Cell cycle; E2F1; Uveal melanoma; miR-181.

Fig. 1

The conservation and cell cycle analysis of miR-181 family members. a Schematic of the miR-181 family putative target sites in the human 3’-UTR of CTDSPL. The sequences of the miR-181 family members are partly evolutionarily conserved in Homo sapiens, Mus musculus, Rattus norvegicus, Bos taurus and Pan troglodytes. Yellow indicates the conserved sequence. The hollow white rectangle indicates the five different gene loci of the miR-181 family members. b and (c) The cell cycle distribution was detected. The fraction of cells in G0/G1-phase was significantly decreased by 8-12%, and the periods of S-phases were significantly increased 6-15% after the mimics of miR-181 family members were transfected into MUM2b cells compared with the control and miR-NC groups (P < 0.05). In contrast, the fraction of cells in G0/G1 phase was significantly increased by 15-18%, and the period of S-phases was significantly decreased 15-20% after the inhibitors of miR-181 family members were transfected into MUM2b cells. d and (e) The fraction of cells in G0/G1-phase was significantly decreased by 10-20%, and the periods of S-phases were significantly increased 8-18% after the mimics of miR-181 family members were transfected into OCM1a cells compared with the control and miR-NC groups (P < 0.05). In contrast, the fraction of cells in G0/G1 phase was significantly increased by 12-15%, and the period of S-phases was significantly decreased 8-12% after the inhibitors of miR-181 family members were transfected into OCM1a cells

Fig. 2

CTDSPL is a direct target of miR-181 family members. a Alignment of the seed sites in the human 3’-UTR of CTDSPL gene. The mutated 3’-UTR of CTDSPL is underlined. b and (c) MUM2b cells were transfected with 40 nM miR-NC, miR-181a, -181b, -181c, and -181d or as-miR-181a, -181b, -181c and -181d. Overexpression or knockdown of miR-181 family inhibited or enhanced CTDSPL expression, respectively. The gray level was analyzed by histogram. d and (f) Overexpression or knockdown of miR-181 expression inhibited or enhanced the Renilla luciferase activity, respectively. 293 T cells were cotransfected with 40 nM miR-NC, miR-181a, -181b, -181c, and -181d, or as-miR-181a, -181b, -181c, and -181d, and 100 ng of reporter plasmid containing the wild-type 3’-UTR of CTDSPL. After 24 h, Renilla luciferase values, normalized against firefly luciferase, were measured. e and (g) The Renilla luciferase activity was nearly unchanged after mimics and inhibitors of miR-181 family members were transfected with the mutated 3’-UTR of CTDSPL

Fig. 3

The expression profile of miR-181 in melanoma tissues and UM cells. a Hierarchical clustering analysis of miRNAs that were differentially expressed in melanoma compared with non-tumor samples. Expression values are represented in shades of red and green indicating expression above and below the median expression value across all samples (log scale 2, from − 2 to + 2), respectively. miR-181b1 and miR-181b2 were significantly upregulated in melanoma tissues. b The expression of miRNA-181a-d was measured by qRT-PCR in RPE, OCM1, SP6.5, VUP, OCM1a, MUM2b and 92-1 cells. miR-181b was overexpressed in OCM1, SP6.5, VUP and 92-1 cells by approximately 50-fold and more than 1000-fold in 92-1 cells, while miR-181b was not upregulated in OCM1a or MUM2b cells. miR-181a was upregulated in OCM1, SP6.5, VUP and 92-1 cells by approximately 12-to-20-fold. The expression levels of miR-181c and miR-181d were not upregulated in most UM cell lines, except for a slight increase in miR-181c in OCM1 cells and miR-181d in OCM1a cells, both less than 10-fold. There was no downregulation of any miR-181 family members. Triplicate assays were performed for each sample, and the relative level of each miRNA was normalized to U6 (*P < 0.05)

Fig. 4

miR-181b inhibits cell cycle distribution through CTDSPL and E2F1. a The miR-181b overexpression plasmid was stably transfected into MUM2b and OCM1a cells (MUM2b-over-miR-181b, OCM1a-over-miR-181b), and the plasmid also contained the EGFP tag. Obvious green fluorescence was observed in MUM2b-over-miR-181b and OCM1a-over-miR-181b cells, but not in the control groups (original magnification 100X). b The qRT-PCR results showed significantly higher expression of miR-181b of nearly 500-fold in MUM2b-over-miR-181b and OCM1a-over-miR-181b cells. c and (d) Cell cycle progression was significantly promoted in miR-181b-stably transfected MUM2b and OCM1a cells. The G0/G1 phase was significantly decreased by approximately 19% and 16%, while that the S-phases was significantly increased by about 10% and 18% in MUM2b-over-miR-181b and OCM1a-over-miR-181b cells, respectively, compared with the control group. e CTDSPL expression was significantly inhibited in miR-181b-stably transfected MUM2b and OCM1a cells. f E2F1 expression was significantly overexpressed in miR-181b-transfected MUM2b and OCM1a cells (*P < 0.05)

Fig. 5

Decreased CTDSPL expression promotes cell cycle distribution through pRB and E2F1. a qRT-PCR results showed significantly decreased expression of CTDSPL after transfection with si-CTDSPL-1 and si-CTDSPL-2 in MUM2b and OCM1a cells, respectively. b The cell cycle G0/G1-phase proportion decreased significantly, approximately 43% and 36% for MUM2b cells and 35% and 32% for OCM1a cells after transfection with si-CTDSPL-1 and si-CTDSPL-2, compared with control group which is 65% in MUM2b cells and 64% in OCM1a cells. While the cell cycle S-phase proportion increased from 13% to 21% and 15% in MUM2b cells and from 10% to 22% and 15% in OCM1a cells after transfection with si-CTDSPL-1 and si-CTDSPL-2 seperately (Fig. 5b). c-d CTDSPL expression was significantly inhibited after transfection with si-CTDSPL-1 and si-CTDSPL-2 in MUM2b and OCM1a cells, whereas pRB and E2F1 expression was significantly increased in MUM2b and OCM1a cells after transfection with si-CTDSPL-1 and si-CTDSPL-2. The gray level was analyzed by histogram seperately (*P < 0.05)

Fig. 6

miR-181 targets CTDSPL, which modulates the cell cycle effector E2F1. Schematic representation of the pathway modulated by miR-181 in UM cells progressing through the cell cycle. miR-181 overexpression in UM cells induces progression through the G1/S transition and promotes S-phase entry. Thus, miR-181 induces cell cycle progression by repressing the downstream target CTDSPL, which in turn results in the phosphorylation of RB and an accumulation of the downstream cell cycle effector E2F1