In vivo and in vitro effects of microRNA-27a on proliferation, migration and invasion of breast cancer cells through targeting of SFRP1 gene via Wnt/β-catenin signaling pathway

Abstract

This study aims to explore the effects of microRNA-27a (miR-27a) targeting of SFRP1 on the proliferation, migration and invasion of breast cancer (BC) cells through the regulation of Wnt/β-catenin signaling pathway. BC and normal breast tissues were obtained from 396 female BC patients and 308 female patients with benign breast lesions respectively. Human normal mammary epithelial (MCF-10A) and BC cell lines (BT-20, MCF-7, T-47D and MDA-MB-231) were cultured. After cell transfection, BC cells were assigned to six groups: control, miR-27a mimics, miR-27a inhibitors, negative control (NC), si-SFRP1 and si-SFRP1 + miR-27a inhibitors groups. qRT-PCR assay and Western blot were employed to detect the expressions of miR-27a, SFRP1, Wnt, β-catenin and GSK3β. MTT assay, wound-healing test and Transwell assay were used to test cell proliferation, migration and invasion. BC tissues were found to have higher miR-27a expression and lower SFRP1 mRNA and protein expressions than MCF-10A cells and normal breast tissues. Compared with the control and NC groups, the miR-27a mimics and si-SFRP1 groups exhibited down-regulation of SFRP1, up-regulation of Wnt, β-catenin and GSK3β, and promotion of cell proliferation, migration and invasion. The miR-27a inhibitor group showed up-regulation of SFRP1 and inhibition of cell proliferation, migration and invasion in comparison to the miR-27a mimic group. The si-SFRP1 + miR-27a inhibitors group also exhibited up-regulation of SFRP1 and inhibition of cell proliferation, migration and invasion in comparison to the si-SFRP1 group. miR-27a may activate the Wnt/β-catenin signaling pathway by negatively regulating SFRP1 to promote the proliferation, migration and invasion of BC cells.

Keywords: Wnt/β-catenin signaling pathway; breast cancer; microRNA-27a; proliferation; secreted frizzled-related protein 1.

Figure 1. The expression of miR-27a in normal breast and BC tissues as detected by qRT-PCR

Note: normal breast tissues, n = 308; BC tissues, n = 396. miR-27a, microRNA-27a; BC, breast cancer; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 2. The expression of miR-27a in normal mammary epithelial and BC cell lines as detected by qRT-PCR

Note: miR-27a, microRNA-27a; BC, breast cancer; qRT-PCR, quantitative real-time polymerase chain reaction; *, compared with T-47D cell line, P < 0.05 (MCF10A cells vs. T-47D cells, P < 0.001; MDA-MB-231 vs. T-47D cells, P = 0.007; BT-20 cells vs. T-47D cells, P = 0.021; MCF-7 cells vs. T-47D cells, P = 0.003).

Figure 3. The SFRP1 mRNA and protein expressions in normal breast and BC tissues, normal mammary epithelial and BC cell lines

A. comparison of the SFRP1 mRNA expression between normal breast tissues and BC tissues (P = 0.018); B. the SFRP1 protein expression in normal breast and BC tissues as detected by Western blot analysis; C. comparison of SFRP1 protein expression between normal breast and BC tissues (P = 0.029); D. comparison of SFRP1 mRNA expression among normal mammary epithelial and BC cell lines (MCF10A cells vs. T-47D cells, P < 0.001; MCF-7 cells vs. T-47D cells, P = 0.013; BT-20 cells vs. T-47D cells, P = 0.038; MDA-MB-231 vs. T-47D cells, P = 0.020); E. SFRP1 protein expression in normal mammary epithelial and BC cell lines detected by Western blot; F. comparison of SFRP1 protein expression among normal mammary epithelial and BC cell lines (MCF10A cells vs. T-47D cells, P < 0.001; MCF-7 cells vs. T-47D cells, P = 0.017; BT-20 cells vs. T-47D cells, P = 0.042; MDA-MB-231 vs. T-47D cells, P = 0.023). Note: BC, breast cancer; SFRP1, secreted frizzled-related protein 1; *, compared with T-47D cell line, P < 0.05.

Figure 4. Effect of miR-27a on SFRP1 as evaluated by TargetScan online database and dual-luciferase reporter gene assay

A. the binding site of miR-27a to SFRP1-3′UTR predicted by TargetScan online database; B. comparison of luciferase activity among the four groups detected by dual-luciferase reporter gene assay. Notes: miR-27a, microRNA-27a; SFRP1, secreted frizzled-related protein 1; WIT, wild type; MUT, mutant; *, compared with the WIT + mimics group, P < 0.05 (WIT + NC vs. WIT + mimics, P = 0.033; MUT + NC vs. WIT + mimics, P = 0.036; MUT + mimics vs. WIT + mimics, P = 0.028).

Figure 5. Effect of miR-27a on the SFRP1 mRNA and protein expressions among the six groups

A. comparison of SFRP1 mRNA expression among six groups; B. comparison of SFRP1 protein expression among the six groups; C. SFRP1 protein expression among six groups detected by Western blot. Note: miR-27a, microRNA-27a; SFRP1, secreted frizzled-related protein 1; *, compared with the control group, P < 0.05 (the miR-27a mimics group vs. the control group, P = 0.029 for SFRP1 mRNA, P = 0.036 for SFRP1 protein; the miR-27a inhibitors group vs. the control group, P = 0.044 for SFRP1 mRNA, P = 0.031 for SFRP1 protein; the si-SFRP1 group vs. the control group, P = 0.021 for SFRP1 mRNA, P = 0.022 for SFRP1 protein); #, compared with the si-SFRP1 group, P < 0.05 (the miR-27a inhibitors + si-SFRP1 group vs. the si-SFRP1 group, P = 0.023 for SFRP1 mRNA, P < 0.001 for SFRP1 protein).

Figure 6. Effect of miR-27a on the proliferation of BC cells among the six groups

Note: miR-27a, microRNA-27a; BC, breast cancer; *, compared with the control group, P < 0.05 (the miR-27a mimics group vs. the control group, P = 0.032; the miR-27a inhibitors group vs. the control group, P = 0.041; the si-SFRP1 group vs. the control group, P = 0.046); #, compared with the si-SFRP1 group, P < 0.05 (the miR-27a inhibitors + si-SFRP1 group vs. the si-SFRP1 group, P = 0.045).

Figure 7. Effect of miR-27a on the migration and invasion of BC cells among the six groups

A. effect of miR-27a on the migration of BC cells among the six groups; B. comparison of wound healing rate among the six groups; C. effect of miR-27a on the invasion of BC cells among the six groups; D. comparison of invasion cell number of BC into Transwell among the six groups. Note: miR-27a, microRNA-27a; BC, breast cancer; *, compared with the control group, P < 0.05 (the miR-27a mimics group vs. the control group, P = 0.041 for wound-healing rate, P = 0.029 for invasive cell number; the miR-27a inhibitors group vs. the control group, P = 0.034 for wound-healing rate, P = 0.028 for invasive cell number; the si-SFRP1 group vs. the control group, P = 0.046 for wound-healing rate, P = 0.035 for invasive cell number); #, compared with the si-SFRP1 group, P < 0.05 (the miR-27a inhibitors + si-SFRP1 group vs. the si-SFRP1 group, P = 0.021 for wound-healing rate, P = 0.038 for SFRP1 invasive cell number).

Figure 8. Effect of miR-27a on Wnt/β-catenin signaling pathway through targeting SFRP1

A. comparisons of mRNA expressions of β-catenin, GSK3β and Wnt among the six groups (the miR-27a mimics group vs. the control group, P = 0.019 for β-catenin mRNA, P = 0.026 for GSK3β mRNA, P = 0.037 for Wnt mRNA; the miR-27a inhibitors group vs. the control group, P = 0.011 for β-catenin mRNA, P = 0.017 for GSK3β mRNA, P = 0.016 for Wnt mRNA; the si-SFRP1 group vs. the control group, P = 0.023 for β-catenin mRNA, P = 0.032 for GSK3β mRNA, P = 0.042 for Wnt mRNA; the miR-27a inhibitors + si-SFRP1 group vs. the si-SFRP1 group, P = 0.039 for β-catenin mRNA, P = 0.043 for GSK3β mRNA, P = 0.039 for Wnt mRNA); B. protein expressions of β-catenin, GSK3β and Wnt among the six groups detected by Western blot; C. comparisons of mRNA expressions of β-catenin, GSK3β and Wnt among the six groups (the miR-27a mimics group vs. the control group, P = 0.027 for β-catenin protein, P = 0.035 for GSK3β protein, P = 0.046 for Wnt protein; the miR-27a inhibitors group vs. the control group, P = 0.022 for β-catenin protein, P = 0.038 for GSK3βprotein, P = 0.033 for Wnt protein; the si-SFRP1 group vs. the control group, P = 0.031 for β-catenin protein, P = 0.039 for GSK3β protein, P = 0.040 for Wnt protein; the miR-27a inhibitors + si-SFRP1 group vs. the si-SFRP1 group, P = 0.042 for β-catenin protein, P = 0.046 for GSK3β protein, P = 0.044 for Wnt protein); D. protein expression of β-catenin in cytoplasm and nucleus among the six groups detected by Western blot. Notes: miR-27a, microRNA-27a; SFRP1, secreted frizzled-related protein 1; GSK3β, glycogen synthase kinase-3β; *, compared with the control and NC groups, P < 0.05; #, compared with the si-SFRP1 group, P < 0.05.