LINC02086 promotes cell viability and inhibits cell apoptosis in breast cancer by sponging miR-6757-5p and up-regulating EPHA2

Abstract

Background: Long non-coding RNAs (lncRNAs) are critical regulators in the initiation and progression of breast cancer. Our study aims to characterize the functions of LINC02086 which few published in breast cancer and decipher the downstream molecular mechanisms.

Methods: LINC02086 expression is tested in RNA-seq data from GEPIA database, tumor tissue samples from hospital patients and breast cancer cell lines. LINC02086 was silenced or overexpressed by lenti-virus-mediated shRNAs, or pLVX-Puro plasmids. Luciferase reporter assay and RNA pull-down assay were applied to study interactions between LINC02086, miR-6757-5p and ephrin type-A receptor 2 (EPHA2). LINC02086-silencing MCF-7 cells were injected into mice to establish xenograft animal models.

Results: Using RNA-seq data, tumor tissue samples and breast cancer cells, LINC02086 was consistently found to be up-regulated in breast cancer, and correlated with poorer prognosis. LINC02086 knockdown decreased cell viability, promoted cell apoptosis and suppressed tumor growth. LINC02086 interacted with miR-6757-5p that interacted with EPHA2.LINC02086 expression was negatively correlated with miR-6757-5p expression (r = -0.5698, P < 0.001) but was positively correlated with EPHA2 expression (r = 0.5061, P < 0.001). miR-6757-5p expression was negatively correlated with EPHA2 expression (r = -0.5919, P < 0.001). LINC02086 regulated EPHA2 via miR-6757-5p. miR-6757-5p/EPHA2 axis was a mediator of the effect of LINC02086 on cell viability and apoptosis.

Conclusion: LINC02086 increases cell viability and decreases apoptotic cells in breast cancer by sponging miR-6757-5p to upregulate EPHA2. This study presents LINC02086/miR-6757-5p/EPHA2 axis as promising therapeutic targets for breast cancer intervention.

Keywords: Breast cancer; EPHA2; Luciferase reporter assay; RNA pull-down assay; lncRNAs; miRNAs.

Fig. 1

LINC02086 is up-regulated in breast cancer and associated with prognosis. A expression data of LINC02086 downloaded from GEPIA database; B, LINC02086 levels in 43 paired cancer specimens and adjacent-normal specimens; C, survival time of patients with high and low LINC02086 expression; D, LINC02086 levels in MCF-10A,MCF-7, MDA-MB-231, ZR751, and SKBR3 cells. *P < 0.05, **P < 0.01, ***P < 0.001 vs. normal or MCF-10A

Fig. 2

LINC02086 knockdown decreases cell viability, promotes cell apoptosis and inhibits tumor growth. A, LINC02086 knockdown by shRNAs transfection in MCF-7 cells; B, cell viability of MCF-7 cells transfected with shRNA-1, 2 at different time points; C-D, percentages of apoptotic cells detected by flow cytometry; E, tumor volume of mice models at different time points; F, tumor weight of mice models at day 35; G, LINC02086 levels in tumors harvested from mice models; H, TUNEL positive cells on tumor tissue samples harvested from mice models. Scale bar = 50 μm. **P < 0.01, ***P < 0.001 vs. shNC

Fig. 3

LINC02086 interacts with and negatively correlates with miR-6757-5p. A cytoplasmic/nuclear fractionation of LINC02086 in MCF-7 and MDA-MB-231 cells; B, the binding site between LINC02086 and miR-6757-5p; C, LINC02086 levels in MCF-7 cells transfected with miR-inhibitor or miR-mimic; D, luciferase reporter activity in MCF-7 cells transfected with miR-mimic and wild type or mutant LINC02086. E, miR-6757-5p enrichment with LINC02086 detected by RNA pull down assay. F, LINC02086 levels in MCF-7 cells transfected with miR-inhibitor or miR-mimic; G, miR-6757-5p levels in 43 paired cancer specimens and adjacent-normal specimens; H, correlations of miR-6757-5p levels with LINC02086 levels in cancer specimens. *P < 0.05, **P < 0.01, ***P < 0.001 vs. miR-NC, control probe or normal

Fig. 4

miR-6757-5p inhibitor alleviates the effects of LINC02086 silencing on cell viability and apoptosis. miR-6757-5p inhibitor or mimic is transfected into MCF-7 cells. A, cell viability of MCF-7 cells; B-C, percentages of apoptotic cells detected by flow cytometry. MCF-7 cells are co-transfected with shRNA-1/shNC and miR-inhibitor/miR-NC. D-F cell viability and percentages of apoptotic cells.***P < 0.001 vs.miR-NC or shNC + miR-NC. #P < 0.05, ###P < 0.001 vs. shRNA-1 + miR-NC

Fig. 5

LINC02086 promotes EPHA2 expression via miR-6757-5p. A, the binding site between miR-6757-5p and EPHA2; B, luciferase reporter activity in MCF-7 cells transfected withmiR-mimic/miR-inhibitor and wild type or mutant LINC02086; C-D, EPHA2 mRNA and protein levels in MCF-7 cells transfected with miR-mimic or miR-inhibitor; E, EPHA2 mRNA levels in paired cancer specimens and adjacent-normal specimens; F, correlations between EPHA2 mRNA levels and miR-6757-5p mRNA levels; G-H, EPHA2 mRNA and protein levels in MCF-7 cells co-transfected with shRNA-1/shNC and miR-inhibitor/miR-NC; I, correlations between EPHA2 mRNA levels and LINC02086 levels. *P < 0.05, **P < 0.01, ***P < 0.001 vs.miR-NC, normal or shNC + miR-NC. ##P < 0.01, ###P < 0.001 vs. shRNA-1 + miR-NC