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, 11, 4729-4742
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miR-142-3p Suppresses Uveal Melanoma by Targeting CDC25C, TGFβR1, GNAQ, WASL, and RAC1

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miR-142-3p Suppresses Uveal Melanoma by Targeting CDC25C, TGFβR1, GNAQ, WASL, and RAC1

Dewei Peng et al. Cancer Manag Res.

Abstract

Purpose: Uveal melanoma (UM) is the most frequent metastatic ocular tumor in adults. Therapeutic intervention remains ineffective since none of the novel procedures used to treat this disease increased survival rates. To deal with this limitation, additional studies are required to clarify its pathogenesis. The current study focused on describing how epigenetic modulation by miR-142-3p affects changes in some cellular functions underlying UM pathogenesis. Methods and results: Microarray analysis identified 374 miRNAs which were differentially expressed between UM cells and uveal melanocytes. miR-142-3p was one of the 10 most downregulated miRNAs. Quantitative RT-PCR analysis confirmed that miR-142-3p expression levels were significantly decreased in both UM cell lines and clinical specimens. The results of the MTS, clone formation, scratch wound, transwell assays, and in vivo biofluorescence imaging showed that miR-142-3p overexpression significantly inhibited cell proliferation, migration, and invasiveness. Nevertheless, miR-142-3p did not affect cell apoptotic activity or sensitivity to doxorubicin. Cell cycle and EdU analysis showed that miR-142-3p overexpression induced G1/G2 cell cycle arrest and reduced DNA synthesis in UM cells. Microarray analysis showed that miR-142-3p mainly regulates the TGFβ signaling pathway, and those in which MAPK and PI3K-Akt are constituents. Functional interactions between miR-142-3p and CDC25C, TGFβR1, GNAQ, WASL, and RAC1 target genes were confirmed based on the results of the luciferase reporter assay and Western blot analysis. CDC25C or RAC1 downregulation is in agreement with cell cycle arrest and DNA synthesis disorder induction, while downregulation of TGFβR1, GNAQ, WASL, or RAC1 accounts for declines in cell migration. Conclusion: miR-143-3p is a potential therapeutic target to treat UM since overriding its declines in expression that occur in this disease reversed the pathogenesis of this disease. Such insight reveals novel biomarker for decreasing UM vitality and for improved tracking of tumor progression.

Keywords: miR-142-3p; molecular mechanisms; tumor suppressor; uveal melanoma.

Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
MiR-142-3p is downregulated in uveal melanoma (UM) cells and clinical specimens. (A) The 20 most upregulated and downregulated miRNAs between UM cells (M17 and SP6.5) and normal uveal melanocytes (D78) were hierarchically clustered and displayed in a heat map. MiR-142-3p was among the most downregulated miRNAs (in red font). (B) RT-qPCR analysis of miR-142-3p levels in UM cell lines (SP6.5 and M17) and uveal melanocytes (D78 and um95). (C) RT-qPCR analysis of miR-142-3p levels in UM tissues and adjacent tissues. 1–20: clinical samples 1–20 of UM and adjacent tissues. Each experiment was carried out three times in triplicates. **P<0.01.
Figure 2
Figure 2
MiR-142-3p inhibits uveal melanoma (UM) cell proliferation through cell cycle G1/G2 arrest and impeding DNA synthesis. (A) MTS assays of UM cells transfected with miR-142-3p or negative control (NC) (n=3/group). (B and C) Colony formation assay of UM cells transfected with miR-142-3p (n=3/group). (D) Cell cycle analysis of UM cells transfected with miR-142-3p (n=3/group). (E) DNA replication assay performed in UM cells transfected with miR-142-3p. Newly synthesized DNA was labeled with EdU (red) (n=3/group). Scale bars: 100 μm. **P<0.01, ***P<0.001.
Figure 3
Figure 3
Apoptotic activity assay in uveal melanoma (UM) cells transfected with miR-142-3p or NC. (A) Hoechst staining assay of UM cells transfected with miR-142-3p. Scale bars: 20 μm (n=3/group). (B) Annexin-V/PI double-staining assay of UM cells transfected with miR-142-3p. Cells were treated with Annexin V/PI and detected by flow cytometry (n=3/group). (C) Caspase 3/7 activity assay on UM cells transfected with miR-142-3p after doxorubicin treatment (n=3/group). Doxorubicin+, cells were treated with doxorubicin for 48 hrs. **P<0.01, ***P<0.001.
Figure 4
Figure 4
Ectopic expression of miR-142-3p inhibits migratory and permeation activity of uveal melanoma (UM) cells. (A) Wound healing assay of UM cells transfected with miR-142-3p or NC (n=3/group). (B) Transwell assay of UM cells transfected with miR-142-3p or NC (n=3/group). (C) Matrigel invasion assay of UM cells transfected with miR-142-3p or NC (n=3/group). Cell permeation was assayed in transwells coated with matrigel. *P<0.05, **P<0.01, ***P<0.001.
Figure 5
Figure 5
Overexpression of miR-142-3p suppresses tumor growth in vivo. (A) In vivo biofluorescence imaging of nude mice following suprachoroidal injection of UM cells transfected with miR-142-3p or NC. (B) The quantitative data for the in vivo biofluorescence imaging assay (n=5/group). (C) Representative image of hematoxylin-eosin staining of eyeball sections from nude mice. The tumor tissues (formed by SP6.5-pGL4.17 cells) have been enclosed by a dashed line. *P<0.05, **P<0.01.
Figure 6
Figure 6
Scatter plot analysis and miR-142-3p-target networks based on mRNA expression profiling. (A) Scatter plot analysis of gene expression profiling for uveal melanoma (UM) cells transfected with miR-142-3p or NC oligonucleotide. (B) A network of hsa-miR-142-3p and its predicted targets. The differentially expressed genes that were also the predicted target of hsa-miR-142-3p in several GO terms and pathway terms are selected to construct this network. (C) Western blot analysis of the expression of phospho-Akt (p-Akt), total Akt, phospho-ERK1/2 (p-ERK1/2), and total ERK1/2 in the UM cells transfected with miR-142-3p or NC. Representative data from three independent experiments are shown. **P<0.01, ***P<0.001.
Figure 7
Figure 7
CDC25C, TGFβR1, GNAQ, WASL, and RAC1 are targets of miR-142-3p. (A) Luciferase activity of HEK293 cells co-transfected with pLuc-targets 3′ UTR (left) or pLuc-targets 3ʹUTR-Mut (right), Renilla luciferase vector (transfection efficiency control), and miR-142-3p or NC. (B) RT-qPCR analysis of CDC25C, TGFβR1, GNAQ, WASL, or RAC1 mRNA levels in the uveal melanoma (UM) cells and uveal melanocytes. (C) RT-qPCR analysis of CDC25C, TGFβR1, GNAQ, WASL, or RAC1 mRNA levels in UM cells transfected with miR-142-3p or NC. (D) Western blot analysis of the protein levels of CDC25C, TGFβR1, GNAQ, WASL, or RAC1 in the UM cells transfected with miR-142-3p or NC. Each experiment was carried out three times. *P<0.05, **P<0.01, ***P<0.001.
Figure 8
Figure 8
MiR-142-3p targets mediate differential effects on uveal melanoma (UM) cell proliferation and migration. (A) Protein expression of CDC25C, TGFβR1, GNAQ, WASL, or RAC1 was efficiently knocked down by corresponding siRNA. (B) MTS assays of UM cells transfected with siRNA against CDC25C, TGFβR1, GNAQ, WASL, or RAC1 (n=3/group). (C) Transwell assay of UM cells transfected with siRNA against CDC25C, TGFβR1, GNAQ, WASL, or RAC1 (n=3/group). (D) Cell cycle analysis of UM cells transfected with siRNA against CDC25C or RAC1 (n=3/group). (E) DNA replication assay performed in UM cells transfected with siRNA against CDC25C or RAC1. Newly synthesized DNA was labeled with EdU (red) (n=3/group). *P<0.05, **P<0.01, ***P<0.001.

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