MicroRNA-34a suppresses breast cancer cell proliferation and invasion by targeting Notch1

Abstract

MicroRNAs (miRs) have been reported to serve critical roles in the progression of tumors. However, thus far, the role of miR-34a in breast cancer is largely unknown. Therefore, the present study aimed to investigate the expression and role of miR-34a in breast cancer, and to further explore the underlying molecular mechanism. Reverse transcription-quantitative polymerase chain reaction was performed to detect the level of miR-34a in human breast cancer tissues. In addition, the role of miR-34a in MCF-7 breast cancer cells was investigated by performing miR-34a overexpression or downregulation through transfection with miR-34a mimic or inhibitor, respectively. Next, the viability and invasion of the MCF-7 cells were respectively analyzed by MTT assay and transwell assay, while apoptosis and cell cycle progression were examined by flow cytometry. Furthermore, associated protein levels were measured using western blotting. The results demonstrated that miR-34a was downregulated in human breast cancer tissues in comparison with the adjacent normal tissues. In addition, Notch1 was demonstrated to be a direct target of miR-34a. miR-34a mimic transfection inhibited MCF-7 cell viability, induced cell apoptosis and G1 phase arrest, and prevented cell invasion, while miR-34a inhibitor transfection resulted in the opposite effects. In conclusion, the presented data indicated that miR-34a suppressed breast cancer cell proliferation and invasion, and its effect may partly be exerted by targeting Notch1.

Keywords: Notch1; breast cancer; invasion; microRNA-34a; proliferation.

Figure 1.

miR-34a expression level in different groups, as detected by reverse transcription-quantitative polymerase chain reaction. Relative expression level of miR-34a in the (A) MCF-10A and MCF-7 cells, (B) MCF-7 cells transfected with miR-34a mimic, mimic control, miR-34a inhibitor or inhibitor control and (C) breast cancer and adjacent normal tissues. **P<0.01 vs. NC group. miR, microRNA; NC, negative control.

Figure 2.

miR-34a directly targets Notch1. (A) Interaction between miR-34a and 3′UTR of Notch1 was predicted by TargetScan. (B) Luciferase activity of a reporter containing a Notch1 3′UTR-WT or 3′UTR-MUT (with a mutation in the miR-34a binding site). (C) Western blots and (D) quantified expression of Notch1 protein in MCF-7 cells in different groups, expressed as the fold of the NC group. (E) Relative mRNA expression level of Notch1 in MCF-7 cells in different groups. *P<0.05 and **P<0.01 vs. NC group. miR, microRNA; NC, negative control; UTR, untranslated region; WT, wild-type; MUT, mutated.

Figure 3.

miR-34a inhibits MCF-7 cell viability. At 48 h after cell transfection, the viability of MCF-7 cells was analyzed by an MTT assay. *P<0.05 and **P<0.01 vs. NC group. miR, microRNA; NC, negative control; OD, optical density.

Figure 4.

miR-34a induces MCF-7 cell apoptosis and G1 phase arrest. At 48 h after cell transfection, the cell cycle distribution and apoptosis of MCF-7 cells were determined by flow cytometry. (A) Cell cycle distribution and (B) the percentage of cells in G1 phase. (C) Apoptosis of MCF-7 cells in different groups. Q2 and Q3 indicate the early and late apoptosis, respectively. (D) The percentage of apoptotic cells. *P<0.05 and **P<0.01 vs. NC group. miR, microRNA; NC, negative control.

Figure 5.

miR-34a prevents MCF-7 cell invasion. At 48 h after cell transfection, the invasion ability of MCF-7 cells was determined by a cell invasion assay, and the data were analyzed. **P<0.01 vs. NC group. miR, microRNA; NC, negative control.