MicroRNA‑124 suppresses cell proliferation and invasion of triple negative breast cancer cells by targeting STAT3

Abstract

MicroRNAs (miRNAs) are pivotal regulators of the progression of carcinogenesis and negatively regulate the expression of tumour‑associated genes. Downregulation of miR‑124 expression has been demonstrated in various human cancer tissues, wherein miR‑124 serves as a tumour suppressor by targeting oncogenes. However, its function and underlying mechanism of action remain unclear in breast cancer. In the present study, the tissue‑specific expression of miR‑124 was detected in 10 paired triple‑negative breast cancer and normal tissues, and its inhibitory effects on cell growth and invasion were evaluated in vitro and in vivo. Bioinformatics analysis identified signal transducer and activator of transcription 3 (STAT3), a well‑known oncogene in breast cancer, as the potential target. Upregulation of miR‑124 expression decreased STAT3 mRNA and protein levels in breast cancer cells and the relative luciferase activity. Rescue experiments revealed that the transfection of a STAT3 expression plasmid reversed the inhibitory effect of miR‑124 on the proliferation and invasion of MDA‑MB‑468 cells. These data demonstrate that miR‑124 serves vital roles in the suppression of triple‑negative breast cancer via inhibition of cell proliferation and invasion through STAT3. These results highlight the potential role of miR‑124 as a diagnostic or therapeutic target in patients with breast cancer.

Figure 1.

miR-124 is downregulated in breast cancer and suppresses cell viability and invasion in vitro. (A) RT-qPCR was performed to evaluate the expression levels of miR-124 in 20 human breast tissue samples. (B) The efficiency of manual upregulation of miR-124 was determined by RT-qPCR in MDA-MB-468 and MDA-MB-231 cells. (C) Cells were transfected with miR-124 and their viability was measured by MTT assay 48 h later (D) miR-124 overexpression lead to compromised invasive capacity of breast cancer cells. All data are presented as mean ± standard deviation of at least three independent experiments. *P<0.05 and **P<0.01 vs. control. miR-124, microRNA-124; NC, negative control; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

Figure 2.

Inhibition of miR-124 promotes cell viability and cell invasion in breast cancer cell lines. (A) Reverse transcription quantitative polymerase chain reaction was performed to evaluate the transfection efficiency of inh-124 (B) Cell viability was measured by MTT assay 2 days post-transfection. (C) Suppressed inh-124 led to enhanced cell invasion in breast cancer cell lines. All data are presented as mean ± standard deviation of at least three independent experiments. *P<0.05 and **P<0.01 vs. control. miR-124, microRNA-124; inh, inhibitor; NC, negative control.

Figure 3.

STAT3 may be a downstream of miR-124 in breast cancer. (A) The potential miR-124 binding area in the WT 3′UTR of STAT3 mRNA, the corresponding MUT sequence is in red. (B) Reverse transcription quantitative polymerase chain reaction was performed to measure the expression of miR-124 in human breast tissues. (C) The correlation between expression of miR-124 and STAT3 in 20 human breast tissue samples. **P<0.01 vs. control. STAT3, signal transducer and activator of transcription 3; UTR, untranslated region; miR-124, microRNA-124; WT, wide type; MUT, mutated.

Figure 4.

miR-124 negatively regulates the expression of STAT3 by direct interaction in breast cancer. (A) The expression of STAT3 mRNA was decreased by overexpression of miR-124 in MDA-MB-468 and MDA-MB-231 cells, while inhibition of miR-124 led to the upregulation of STAT3. (B) The corresponding western blot data of STAT3. Data values represent the relative intensity values. (C) A dual luciferase assay was performed following co-transfection with WT or MUT reporters and miR-124 or miR-NC in MDA-MB-468 cells. miR-124 overexpression lead to a significant decrease in the activity of the WT reporter but not the MUT reporter. Firefly luciferase activity was used to normalize the data. All data are presented as mean ± standard deviation of at least three independent experiments. *P<0.05 and **P<0.01 vs. control. miR-124, microRNA-124; STAT3, signal transducer and activator of transcription 3; inh, inhibitor; miR-124, microRNA-124; WT, wild type; MUT, mutant; NC, negative control.

Figure 5.

Restoration of STAT3 impairs the prohibitive effects of miR-124 on cell viability and invasion in MDA-MB-468. (A) miR-124 expression levels in MDA-MB-468 cells infected with LV-miR-124. (B) Stable expression of miR-124 suppressed the expression of STAT3. However, STAT3 mRNA and protein levels were restored by transfection with plasmid-STAT3. Data values represent the relative intensity value. (C) STAT3 overexpression reversed the prohibitive cell viability caused by LV-miR-124. (D) A significantly suppressed invasive capacity was detected in MDA-MB-468 cells subsequent to LV-miR-124 transfection. This was partially recovered following plasmid-STAT3 transfection. All data are presented as mean ± standard deviation of at least three independent experiments. **P<0.01 vs. control. STAT3, signal transducer and activator of transcription 3; miR-124, microRNA-124; LV-miR-124, miR-124 expression lentivirus; plasmid-STAT3, STAT3 expression plasmid; NC, negative control.

Figure 6.

Transfection efficiency of plasmid-STAT3 and LV-si-STAT3 in MDA-MB-468 cells. (A) The transfection efficiency of plasmid-STAT3 was measured using RT-qPCR assays and western blot analysis. (B) RT-qPCR assays and western blot analysis were performed to evaluate the transfection efficiency of LV-si-STAT3. All data are presented as mean ± standard deviation of at least three independent experiments. Data values included in the western blot gel images represent the relative intensity value of the bands. **P<0.01 vs. control. STAT3, signal transducer and activator of transcription 3; siRNA, small interfering RNA; LV-si-STAT3, si-STAT3 lentivirus; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

Figure 7.

Overexpression of miR-124 and downregulation of STAT3 lead to smaller tumor sizes in vivo. (A) The proliferation curves of MDA-MB-468 cells following stable transfection of miR-124 or LV-si-STAT3 were obtained after 5 days of observation. (B) The representative nude mice and corresponding tumors for the four groups at 3 weeks post-inoculation. *P<0.05 and **P<0.01 vs. control. miR-124, microRNA-124; STAT3, signal transducer and activator of transcription 3; siRNA, small interfering RNA; LV-si-STAT3, si-STAT3 lentivirus.