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. 2020 Jan-Dec:19:1533033820980098.
doi: 10.1177/1533033820980098.

microRNA-214 Prevents Traits of Cutaneous Squamous Cell Carcinoma via VEGFA and Bcl-2

Xianpeng Ma  1 , Di Wu  2 , Xiaodong Zhang  1 , Xiao Shao  3 , Guangyao Hu  4
Affiliations

microRNA-214 Prevents Traits of Cutaneous Squamous Cell Carcinoma via VEGFA and Bcl-2

Xianpeng Ma et al. Technol Cancer Res Treat. 2020 Jan-Dec.

Abstract

Background: Dysregulation of microRNA-214 (miR-214) has been indicated in different tumors. The function of miR-214 in cutaneous squamous cell carcinoma (CSCC) is yet to be deciphered. The current study aimed to investigate the specific mechanism underpinning CSCC development with the involvement of miR-214 and its putative targets.

Methods: Microarray analysis of CSCC and adjacent tissues was carried out to filter the most significant downregulated miRNA. Survival analysis of patients was subsequently implemented, followed by miRNA expression determination in CSCC cells. Gain-of-function assays were performed to evaluate its function on cellular level. The targets of the determined miRNA were predicted and their expression in CSCC and adjacent tissues was evaluated. The targeting relationship was analyzed by dual-luciferase assays. Finally, rescue experiments were conducted.

Results: miR-214 was reduced in CSCC tissues and cells, and the survival of patients harboring overexpression of miR-214 was higher. miR-214 restoration increased CSCC cell apoptosis, while decreased proliferative, invasive and migratory activities. miR-214 interacted with vascular endothelial growth factor A (VEGFA) and B-cell CLL/lymphoma 2 (Bcl-2). VEGFA and Bcl-2, overexpressed in CSCC tissues and cells, were negatively correlated with miR-214. Moreover, VEGFA and Bcl-2 overexpression reversed the anti-tumor phenotypes of miR-214 on CSCC cells. miR-214 disrupted the Wnt/β-catenin pathway through VEGFA and Bcl-2 in the CSCC cells.

Conclusion: Our data demonstrates that miR-214 exerts a suppressing role in CSCC. The discovery of novel targets such as miR-214 and VEGFA/Bcl-2 may facilitate the development of therapeutic options.

Keywords: Bcl-2; Cutaneous squamous cell carcinoma; The Wnt/β-catenin pathway; VEGFA; microRNA-214.

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Conflict of interest statement

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
miR-214 is screened out to be significantly reduced in CSCC cells. A, detection of differentially expressed miRNAs by microarray analysis; B, RT-qPCR detection of miR-214 expression in cancer and adjacent tissues (*p < 0.05 according to the 2-way ANOVA); C, survival analysis of CSCC patients with different miR-214 expression; D, RT-qPCR detection of miR-214 expression in cancer and HaCaT cells (*p < 0.05 according to the 1-way ANOVA); E, RT-qPCR detection of miR-214 expression in CSCC cells overexpressing miR-214 (*p < 0.05 according to the 2-way ANOVA).
Figure 2.
Figure 2.
miR-214 overexpression retards the growth, migration and invasion capacities of CSCC cells. CSCC cells were transfected with miR-214 mimic or control. A, CSCC cell viability evaluated by CCK-8 assay (*p < 0.05 according to the 2-way ANOVA); B, EdU detection of CSCC cell proliferative activity (*p < 0.05 according to the 2-way ANOVA); C, changes of apoptosis ability of CSCC cells detected by flow cytometry (*p < 0.05 according to the 2-way ANOVA); D, CSCC cell migration evaluated by Transwell assay (*p < 0.05 according to the 2-way ANOVA); E, CSCC cell invasion evaluated by Transwell assay (*p < 0.05 according to the 2-way ANOVA).
Figure 3.
Figure 3.
miR-214 directly targets VEGFA and Bcl-2. A, genes targeted by miR-214 screened out by StarBase, miRWalk, miRBase and Targetscan websites; B, RT-qPCR detection of expression of each gene in cancer tissues and adjacent tissues (*p < 0.05 according to the 2-way ANOVA); C, correlation analysis of miR-214 and VEGFA; D, correlation analysis of miR-214 and Bcl-2; E, targeting relationship between miR-214 and VEGFA/Bcl-2 (*p < 0.05 according to the 2-way ANOVA); F, RT-qPCR detection of VEGFA and Bcl-2 expression in CSCC cells and normal cells (*p < 0.05 according to the 2-way ANOVA); G, survival analysis of CSCC patients with different VEGFA expression; H, survival analysis of CSCC patients with different Bcl-2 expression; I, RT-qPCR detection of expression of VEGFA and Bcl-2 after overexpression of miR-214 (*p < 0.05 according to the 2-way ANOVA).
Figure 4.
Figure 4.
Overexpression of VEGFA or Bcl-2 restores viability of CSCC cells. CSCC cells were transfected with VEGFA OE, Bcl-2 OE or NC in the presence of miR-214 mimic. A, RT-qPCR detection of expression of VEGFA and Bcl-2 after co-transfection (*p < 0.05 according to the 2-way ANOVA); B, CSCC cell viability evaluated by CCK-8 assay (*p < 0.05 according to the 2-way ANOVA); C, EdU detection of CSCC cell proliferative activity (*p < 0.05 according to the 2-way ANOVA); D, changes of apoptosis ability of CSCC cells detected by flow cytometry (*p < 0.05 according to the 2-way ANOVA); E, CSCC cell migration evaluated by Transwell assay (*p < 0.05 according to the 2-way ANOVA); F, CSCC cell invasion evaluated by Transwell assay (*p < 0.05 according to the 2-way ANOVA).
Figure 5.
Figure 5.
miR-214 impairs the Wnt/βcatenin pathway by interacting with VEGFA/Bcl-2. The protein expression of βcatenin and Cyclin D1 in CSCCs transfected with miR-214 alone or with VEGFA OE/Bcl-2 OE assessed by western blot (*p < 0.05 vs. miR-214 control treatment, #p < 0.05 vs. miR-214 mimic + NC treatment according to the 2-way ANOVA).
Figure 6.
Figure 6.
A schematic representation of the miR-214-VEGFA/Bcl-2-Wnt/βcatenin cascade in CSCC cells. miR-214 bound to VEGFA and Bcl-2 in a direct manner and inhibited their transcription, and VEGFA and Bcl-2 affect the Wnt/β-catenin pathway activation, thus influencing proliferation and apoptosis abilities.
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