Background and Aims: Runt-related transcription factor 2 (RUNX2) and its target gene matrix metalloproteinase 3 (MMP-3) are highly expressed in breast cancer tissue and related to tumor metastasis, but whether RUNX2 can affect the invasion and metastasis of breast cancer cells by regulating MMP-3 gene transcription is unclear. This study was conducted to analyze the relationship between RUNX2 and MMP-3 mRNA levels in breast cancer tissues by bioinformatics approaches, and observe the effect of RUNX2 expression on the transcription level of MMP-3 gene and the invasion ability in breast cancer cells.  
Methods: The mRNA expression data of RUNX2 and MMP-3 as well as the relevant survival data in 1 092 breast cancer patients were obtained through cBioPortal database, and the correlation between RUNX2 and MMP-3 mRNA levels was analyzed. According to the median of RUNX2 and MMP-3 mRNA levels, the patients were divided into high and low RUNX2 or MMP-3 expression groups, and the association of RUNX2 and MMP-3 expression levels with the distant metastasis-free survival (DMFS) of the patients were analyzed by Kaplan Meier Plotter. The breast cancer MDA-MB-231 and BT-549 cells were transfected with RUNX2 siRNA or RUNX2 overexpression plasmids respectively, and then, the mRNA and protein expression levels were determined by qRT-PCR and Western blot. the potential binding site of RUNX2 in MMP-3 promoter was predicted by bioinformatics software, and then was verified in vitro by ChIP-PCR and double luciferase report system. The relations of RUNX2 and MMP-3 expression levels with the invasion ability of breast cancer cells were analyzed in MDA-MB-231 and BT-549 cells transfected with RUNX2 overexpression plasmid and MMP-3 siRNA by Transwell assay.
Results: There was a positive correlation between the mRNA levels of MMP-3 and RUNX2 in breast cancer (r=0.304 2, P<0.000 1). The DMFS in patients with high RUNX2 expression or high MMP-3 expression was lower than in patients with the corresponding low expression (P=0.034, P=0.013). In MDA-MB-231 cells, both mRNA transcription level and protein level of MMP-3 were decreased after down-regulating RUNX2 expression; and the MMP-3 mRNA and protein levels were increased in MDA-MB-231 and BT-549 cells after up-regulating RUNX2 expression. Results of bioinformatics analysis show that there was a possible binding site of RUNX2 in MMP-3 promoter sequence (5'-ACCACA-3'), and direct binding of RUNX2 to MMP-3 gene promoter region was confirmed by ChIP-PCR and double luciferase report system. The invasion abilities of both MDA-MB-231 and BT-549 cells were significantly increased after RUNX2 overexpression, and these effects were abolished by simultaneous down-regulation of MMP-3 expression (all P<0.05). 
Conclusion: RUNX2 enhances the invasion ability of breast cancer cells possibly by regulating the transcription of MMP-3 gene, and the patients with high RUNX2 and MMP-3 expressions may face a poor prognosis. RUNX2 may be a potential target for the treatment of breast cancer.