MiR-27a Facilitates Breast Cancer Progression via GSK-3β

Abstract

Breast cancer remains one of the leading causes of cancer-associated death in women. MiR-27a is highly expressed in breast cancer tissue. However, the underlying mechanisms that promote breast cancer progression are unknown. In this study, we investigated the regulatory mechanisms of miR-27a and its target glycogen Synthase Kinase 3-β (GSK-3β) in breast cancer cells. We found that miR-27a was highly expressed in breast cancer tissues, which downregulated GSK-3β expression. We further identified GSK-3β as a direct target of miR-27a, and found that the miR-27a mediated suppression of GSK-3β activated Wnt/β-catenin-associated proliferative and invasive factor in breast cancer. The cell transfection assay demonstrated the overexpression of miR-27a also enhanced cell proliferation and invasion, and reduced cell apoptosis through GSK-3β. Finally, we demonstrated that the overexpression of miR-27a facilitated breast cancer progression through its ability to down-regulate the phosphorylation of GSK-3β both in vivo and vitro. These findings highlighted miR-27a as a novel therapeutic target in breast cancer.

Keywords: GSK-3β; breast cancer; miR-27a; phosphorylation; therapeutic target.

Figure 1.

Expression of miR-27a and GSK-3β in breast cancer tissue (A) MiR-27a and GSK-3β expression in breast cancer tissues and para-carcinoma tissues detected by RT-qPCR and Western blot analysis, respectively. *P < 0.05 vs. para-carcinoma tissues. Statistical data were analyzed using independent-samples T test. (B) Expression of miR-27a and GSK-3β in breast cancer tissues according to TNM stage determined through RT-qPCR and Western blot analysis, respectively. *p < 0.05 vs. Stage 1 groups, #p < 0.05 vs. Stage 2 groups, @p < 0.05 vs. Stage 3 groups. Statistical data were analyzed using one-way ANOVA test. (C) Correlation between miR-27a and p-GSK-3β/GSK-3β expression in para-carcinoma and breast cancer tissues (Left, para-carcinoma tissues; Right, breast cancer tissues) assessed by the Pearson correlation test which demonstrated that miR-27a expression inversely correlated with the expression of GSK-3β in breast cancer tissues. ***P < 0.001.

Figure 2.

Expression of miR-27a and GSK-3β in breast cancer cell lines. (A-B) Expression of miR-27a and GSK-3β in breast cancer cell lines evaluated by RT-qPCR and Western blot analysis, respectively. ^*P < 0.05 vs. the MCF-10A group. Statistical data were analyzed using one-way ANOVA test. (C) Binding sequences between miR-27a and GSK-3β assessed by luciferase reporter assays. Statistical data were analyzed using 2-way ANOVA test *p < 0.05 vs. mimic NC groups.

Figure 3.

MiR-27a overexpression promotes cell proliferation through the regulation of GSK-3β in BT-20 cells. (A) Expression of miR-27a in NC mimic, miR-27a mimic, NC mimic+vector, and miR-27a+GSK-3β groups confirmed by RT-qPCR. (B) Expression of p-GSK-3β, GSK-3β, β-catenin, c-myc, and cyclin D1 in NC mimic, miR-27a mimic, NC mimic+vector, and miR-27a+GSK-3β groups measured by Western blot analysis. The corresponding statistic graphics are shown (left bottom and top right corner). (C) Proliferative ability of BT-20 cells in NC mimic, miR-27a mimic, NC mimic+vector, and miR-27a+GSK-3β groups examined via MTT assays. *p < 0.05 vs. the NC mimic group, #p < 0.05 vs. the miR-27a mimic group. Statistical data from (A), (B), (C) were analyzed using one-way ANOVA test.

Figure 4.

MiR-27a overexpression suppresses cell apoptosis and promotes cell invasion through the regulation of GSK-3β in BT-20 cells. (A) Apoptosis rates of BT-20 cells in NC mimic, miR-27a mimic, NC mimic+vector, and miR-27a+GSK-3β groups determined by flow cytometry. (B) Invasion ability of BT-20 cells in NC mimic, miR-27a mimic, NC mimic+vector, and miR-27a+GSK-3β groups assessed via transwell assays. Statistical data were analyzed using one-way ANOVA test. *p < 0.05 vs. NC mimic group, #p < 0.05 vs. miR-27a mimic group, Scale bar = 50μm.

Figure 5.

Up-regulated miR-27a transfected in BT-20 cells promotes tumor growth through the regulation of GSK-3β in vivo. (A) Tumor weights were measured in NC mimic, miR-27a mimic, NC mimic+vector, and miR-27a+GSK-3β groups. Statistical data were analyzed using one-way ANOVA test. (B) Survival rates of mice in NC mimic, miR-27a mimic, NC mimic+vector, and miR-27a+GSK-3β groups through Kaplan-Meier analysis, which demonstrated that GSK-3β overexpression combined with miR-27a mimic improved the survival rate, compared with miR-27a mimic treatment alone. The relationship between different treatment groups and survival percentage analyzed by Kaplan-Meier. (C) p-GSK-3β, GSK-3β, β-catenin, c-myc, and cyclin D1 levels in NC mimic, miR-27a mimic, NC mimic+vector, and miR-27a+GSK-3β groups assessed by Western blot analysis. Statistical data were analyzed using one-way ANOVA test. The graphics closed to panel C were corresponding statistic graphics. *p < 0.05 vs. NC mimic groups, #p < 0.05 vs. miR-27a mimic groups.