MiR-135-5p inhibits TGF-β-induced epithelial-mesenchymal transition and metastasis by targeting SMAD3 in breast cancer

Abstract

Breast cancer (BC) is the most frequently diagnosed malignant tumors and the leading cause of death due to cancer in women around the world. A growing body of studies have documented that microRNA (miR)-135-5p is associated with the development and progression of BC. Considering that sekelsky mothers against dpp3 (SMAD3) plays a crucial role in transforming growth factor (TGF)-β/SMAD pathway and epithelial-mesenchymal transition (EMT) process, it is critical to elucidate the crosstalk and underlying regulatory mechanisms between miR-135-5p and SMAD3 in controlling TGF-β-mediated EMT in BC metastasis. Our results revealed a reciprocal expression pattern between miR-135-5p and SMAD3 mRNA in BC tissues and cell lines. Moreover, miR-135-5p was decreased in BC tissues compared to adjacent breast tissues; more interesting, miR-135-5p mRNA levels (Tumor/Normal, T/N) was further decreased in BC patients with lymph node metastasis, while SMAD3 mRNA levels were increased. Gain- and loss-of-function assays indicated that overexpression of miR-135-5p inhibited TGF-β-mediated EMT and BC metastasis in vitro and in vivo. Furthermore, knockdown of SMAD3 produced a consistent phenotype of miR-135-5p overexpression in breast cancer cells. Mechanistically, SMAD3, a pivotal transcriptional modulator of TGF-β/SMAD pathway, for the first time, was analyzed and identified as a target gene of miR-135-5p by bioinformatic algorithms and dual-luciferase reporter assays. Taken together, we clarified that miR-135-5p suppressed TGF-β-mediated EMT and BC metastasis by negatively regulating SMAD3 and TGF-β/SMAD signaling. Our findings supported that miR-135-5p may serve as a tumor suppressor, and be a valuable diagnostic biomarker for the treatment of BC.

Keywords: SMAD3; breast cancer; epithelial-to-mesenchymal transition; metastasis.; microRNA-135-5p.

Figure 1

miR-135-5p expression is decreased and negatively associated with SMAD3 in BC tissues and cell lines. (A and B) The differential expression of miR-135-5p and SMAD3 between 66 paired BC T and N tissues (left panel). The log10 transformed expression levels (T/N) of miR-135-5p and SMAD3 in each sample are shown (right panel). RT-qPCR assays were performed three times. (C) Negative association between the mRNA levels of miR-135-5p and SMAD3 in 66 paired BC tissues. X axis shows the log10 transformed mRNA levels (T/N) of miR-135-5p, and Y axIs represents that of SMAD3. (D) The negative correlation between miR-135-5p and SMAD3 mRNA levels in BC was verified by LinkedOmics dataset. (E and F) miR-135-5p and SMAD3 mRNA levels were repeatedly verified by bioinformatic algorithms Gene Expression Omnibus (GSE19536) and The Cancer Genome Atlas (205398_s_at), respectively. (G) RT-qPCR analysis of the mRNA levels of miR-135-5p and SMAD3 in normal human breast epithelial cells and BC cells. U6 and β-actin were used as internal controls. * P < 0.05, *** P < 0.001.

Figure 2

miR-135-5p negatively regulate SMAD3 by directly targeting 3'-UTR of SMAD3. (A) Schematic flowchart of the subcloning of potential binding sites between miR-135-5p and SMAD3 3'-UTR in psiCHECK-2 luciferase vector. Prediction binding relationship between SMAD3 and the wild-type/mutant of miR-135-5p. (B and C) Results of relative luciferase activities in MDA-MB-231 and MCF-7 cells co-transfected with luciferase reporter constructs (WT or MUT) and miR-135-5p or miR-NC mimics. (D) miR-135-5p expression was determined in MDA-MB-231 cells 48 h post-transfection with miR-135-5p or inhibitors. Then, SMAD3 (E) mRNA and (F) protein levels were analyzed. (G) miR-135-5p expression was evaluated in MCF-7 cells, and SMAD3 (H) mRNA and (I) protein levels were determined. * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 3

miR-135-5p inhibits TGF-β-mediated EMT and cellular invasion by suppressing TGF-β/SMAD pathway. (A and B) Western blot analyze the protein levels of SMAD3, Vimentin and Snail in MDA-MB-231 and MCF-7 cells at 24 h post-transfected with miR-135-5p and negative control, then were stimulated with or without TGF-β1. (C and D) Transwell assays quantify the effect of miR-135-5p on the migratory and invasive ability of MDA-MB-231 cells stimulated with or without TGF-β1 for 24 h. Scale bar, 100 μm. (E and F) The effect of miR-135-5p on the migratory and invasive ability of MCF-7 cells stimulated with or without TGF-β1 for 24 h was determined by Transwell assays. Scale bar, 100 μm. * P < 0.05, ** P < 0.01.

Figure 4

Knockdown of SMAD3 represses TGF-β-mediated EMT and BC cellular invasion. (A) Using RT-qPCR and Western blotting to quantify the mRNA and protein levels of SMAD3 in MDA-MB-231 cells transfected with SMAD3-targeted si-RNAs (B) Western blot analyze the protein levels of SMAD3, Vimentin and Snail in MDA-MB-231 cells at 24 h post-transfected with SMAD3-targeted si-RNAs, then were stimulated with or without TGF-β1. (C and D) Detection of the mRNA levels of SMAD3 and the protein levels of SMAD3, Vimentin and Snail in MCF-7 cells using RT-qRCR and Western blotting, BC cells were treated as above. (E and F) The effect of SMAD3 knockdown on the migratory and invasive ability of MDA-MB-231 cells stimulated with or without TGF-β1 was detected by Transwell. Scale bar, 100 μm. (G and H) Transwell assays quantify the effect of SMAD3 knockdown on the migratory and invasive ability of MCF-7 cells stimulated with or without TGF-β1. Scale bar, 100 μm. * P < 0.05, ** P < 0.01.

Figure 5

miR-135-5p inhibits BC cell metastasis in vivo. (A) Schematic illustration of animal experiments with lung metastases. (B) Using RT-qPCR and Western blotting to quantify the expression of miR-135-5p mRNA (left panel) and SMAD3 protein (right panel) in MDA-MB-231 stable cells. (C) Representative images showing lung metastatic nodules (left panel) in mice injected with MDA-MB-231 cells (LV-miR-13p-5p or LV-miR-NC). Scale bar, 1 cm. Then, H&E assay evaluated the micrometastases (right panel) in the lung sections; Red arrowheads, metastatic nodules and micrometastases. Scale bar, 50 μm. (D and E) Comparison and analysis of the lung metastatic nodules and micrometastases in Fig. 5C. * P < 0.05. (F and G) Kaplan-Meier plotter dataset was utilized to evaluate the prognostic role of SMAD3 and miR-135-5p in the overall survival of breast cancer patients. * P < 0.05.