MicroRNA-34a targets epithelial to mesenchymal transition-inducing transcription factors (EMT-TFs) and inhibits breast cancer cell migration and invasion

Abstract

MicroRNA-34a (miR-34a) plays an essential role against tumorigenesis and progression of cancer metastasis. Here, we analyzed the expression, targets and functional effects of miR-34a on epithelial to mesenchymal transition-inducing transcription factors (EMT-TFs), such as TWIST1, SLUG and ZEB1/2, and an EMT-inducing protein NOTCH1 in breast cancer (BC) cell migration and invasion and its correlation with tumorigenesis and clinical outcomes. Expression of miR-34a is downregulated in human metastatic breast cancers (MBC) compared to normal breast tissues and is negatively correlated with clinicopathological features of MBC patients. Ectopic expression of miR-34a in MBC cell-line BT-549 significantly inhibits cell migration and invasion, but exhibits no clear effect on BC cell growth. We found that miR-34a is able to inactivate EMT signaling pathway with mediatory of NOTCH1, TWIST1, and ZEB1 upon 3'-UTR activity in MBC cell lines, but has no inhibitory effects on SLUG and ZEB2. Furthermore, we investigated the synergistic effects of Thymoquinone (TQ) and miR-34a together on the expression of EMT-associated proteins. Results showed that co-delivery of miR-34a and TQ is able to inactivate EMT signaling pathway by directly targeting TWIST1 and ZEB1 in BT-549 cell line, indicating that they might be a promising therapeutic combination against breast cancer metastasis. Epigenetic inactivation of the EMT-TFs/miR-34a pathway can potentially alter the equilibrium of these regulations, facilitating EMT and metastasis in BC. Altogether, our findings suggest that miR-34a alone could serve as a potential therapeutic agent for MBC, and together with TQ, their therapeutic potential is synergistically enhanced.

Keywords: breast cancer; epithelial-mesenchymal transition; metastasis; microRNA-34a; thymoquinone.

Figure 1. Expression of miR-34a in BC cell lines and BC specimens

A. The expression levels of miR-34a in normal breast significantly decreased in human cancer tissues that measured by TaqMan® MicroRNA assays kit. B. Total RNA was prepared from the cell lysates and expression of miR-34a was quantified by TaqMan MicroRNA Assays. The expressions of miR-34a was normalized against the expression level of U6 snRNA. The expression levels T-47D, BT-549, MDA-MB-231, and MDA-MB-435 were expressed as relative to the miR-34a expression level of MCF-7 cells, non-metastatic BC cell lines (mean±SD). Inverse relationship between levels of miR-34a in 48 human breast specimens with Ki67 C. PR expression D. The diamond indicates human BC specimens (n = 33), and circles represent paired adjacent normal tissue (n = 15). Data were analyzed by Spearman's rank correlation coefficient. In general, it was observed significant correlation between the miR-34a expression levels and some clinicopathological features of MBC patients. Data presented is a representative of three different experiments. (NM, No measured value; * P<0.05).

Figure 2. Ectopic expression of miR-34a inhibits the cell migration and invasion in BT-549 BC cells

A. The BT-549 cells were transfected with plasmid pre-miR-34a and pre-miR-34a-controls as indicated. The miR-34a expression which measured by stem-loop qRT-PCR in transfected cells was significantly increased. B. The relative expression of EMT-TFs in BT-549 cells transfected with miR-34a mimic were analyzed by qRT-PCR. C. Cells were treated the same as above and protein lysis were further analyzed by Western blot assay for the protein expression of EMT-TFs. The over-expressed miR-34a cells reduced NOTCH1, TWIST1, and ZEB1 levels. Nevertheless, the SLUG and ZEB2 levels are no change. D. miR-34a dependently inhibited, migration and invasive characteristics of BT-549 cells. However, no clear effect on cancer cell growth. All data were showed with mean ± SD. Similar results were obtained from three independent experiments. The qRT-PCR results normalized to 18S RNA (18S) or U6 snRNA (U6). In Western blot, the β-actin (ACTIN) as internal controls. (* P<0.05).

Figure 3. EMT-TFs demonstrate high expression levels in MBC cell lines in front of miR-34a expression

Relative expression levels of NOTCH1 A., SLUG B., TWIST1 C., and ZEB1 D. and ZEB2 E. were detected in non-metastatic BC cell lines (MCF7 and T47D), and MBC cell lines (MDA-MB-231, MDA-MB-435 and BT-549), in comparison to relative expression levels of miR-34a using qRT-PCR. F. EMT-TFs expression level was detected in MBC cell lines by western blot. Totally, EMT-TFs were overexpressed in invasive BC cell lines. Similar results were obtained from three independent experiments. Horizontal bars show with mean±SD. The Tubulin (TUB) in western blot assay, and 18S or U6 in qRT-PCR as controls. (* P<0.05 and ** P<0.001 versus MCF-7 group).

Figure 4. EMT-TFs are frequently upregulated in BC tissues

A. SLUG, TWIST1 ZEB1/2 and NOTCH1 expression level were detected in MBC tissues and matched with the corresponding paired adjacent normal tissue through qRT-PCR. B. EMT-TFs are differentially expressed in primary malignant BC tissue compared with the corresponding paired adjacent normal tissue. For each case of the 33 patients, index of all EMT-TFs (SLUG, TWIST1, and ZEB1/2) and NOTCH1, expression (qRT-PCR score) of the primary tissue was subtracted by that of the corresponding paired adjacent normal tissue, and the resultant values (Malignant-NAT) from all 33 cases were used for plotting. The relative of EMT-TFs expression level between primary cancer and paired adjacent tissue of each case were represented as a dot. EMT-TFs are considered to be significantly upregulated, similar or negative in primary tissue when compared with the corresponding paired adjacent tissue only if the calculated index is >0 (circular dots), 0 (square dot), <0 (triangular dots), respectively. C. Kaplan-Meier survival analysis for the relationship between survival time and ZEB1 signature in BC was performed by using the online tool (http://kmplot.com/analysis/). All data were showed with mean ± SD. Data presented is a representative of three different experiments. The qRT-PCR results were normalized to18S. RFS, relapse free survival; OS, overall survival; DMFS, distant metastasis free survival; PPS, post progression survival.

Figure 5. Correlations between relative expression levels of NOTCH1 A., SLUG B., ZEB1 C., and ZEB2 D. with miR-34a expression level

Data indicate significantly negative associations in EMT-TFs and miR-34a expressions in MBC patients. The diamond indicates human BC specimens (n = 33), and circles represent paired adjacent normal tissue (n = 15). Data were analyzed by Spearman's rank correlation coefficient.

Figure 6. TWIST1, NOTCH1 and ZEB1 are direct targets of miR-34a

A. miR-34a binding sequences at the 3′-UTR of NOTCH1, TWIST1 and ZEB1. Mutation was generated in the NOTCH1, and ZEB1 3′UTR sequence in the complementary site for the seed region of miR-34a. BT-549 cells were transfected with either the wild-type or mutant NOTCH1 B., TWIST1 C., ZEB1 D. -3′UTR luciferase reporter genes, together with different concentrations of miR-34a mimic or controls (0, 10, 30, and 90 ng). The relative luciferase values were measured and normalized by luciferase reporter assay after 48 h. NOTCH1, TWIST1, and ZEB1 expression were directly down-regulated by miR-34a. All data were showed with mean ± SD. Similar results were obtained from three independent experiments. (* P<0.05 and ** P<0.001 versus 0 ng treated group).

Figure 7. Synergic effects of thymoquinone (TQ) and miR-34a on mRNA (top panel) and protein (bottom panel) level expression of EMT-TFs

TQ (5 μM) treatment for 6h for RT-PCR or 12h for Western blot pre-miRNAs (20 ng of each) treatment for 36 h inhibited the expression of NOTCH1 A., TWIST1 B., and ZEB1 C. in the BT-549 cell line. Results expressed as mean ± SD, and independently repeated with three times. qRT-PCR results normalized to 18S or U6 and compared with the untreated cells as a control. In western blot β-actin (ACTIN) as an internal control (* P<0.05 and ** P<0.001 versus control group).

Figure 8. Molecular model of BC metastasis suppressed via TQ/miR-34a-initiated signaling pathways

This schematic carton shows the major triggering of EMT-inducing signaling pathways, like Wnt proteins (NOTCH), Ras/MAPK kinas, PI3-kinase/Akt and hypoxia (ROS). Co-delivery of TQ/miR-34a leads to repression of metastatic, cell differentiation, and EMT factures in front of inducing of apoptosis and TP53 activations and MET features of BC cell. For more details see manuscript text. TQ, Thymoquinone; miR-34a, microRNAs-34a; PI3K/AKT, phosphatidylinositol 3-kinase (PI3K)/Akt; ROS, reactive oxygen species; MAPK, Mitogen-Activated Protein Kinase; ZEB, zinc-finger E-box-binding; EMT, epithelial to mesenchymal transitions, MET, mesenchymal to epithelial transitions; EMT-TFs, EMT-inducing transcription factors.