MiRs are supported by accumulating evidence as practical biomarkers for cancer management. Many miRs in different cancers are aberrantly expressed, and the roles of certain miRs have also been described [16, 17]. Genome-wide analysis of miRs with the MiRaGE approach designated that miR-612 was downregulated in cancer tissues comparing to non-tumorous tissues in colorectal cancer . The exact physiological role of miR-612, however, was unknown, especially in cancer development.
Our study revealed that miR-612 has inhibitory functions on cell proliferation, invasion, and migration and enhances apoptosis, and cell cycle arrest of BC cells. Also, we reported that miR-612 expression was inversely associated with BC cell inhibition. We designated that miR-612 significantly decreased the BC cell viability, proliferation, and colony formation ability. These data stipulate that miR-612 suppresses migration, proliferation, and invasion in BC cells, but it is not clear whether miR-612 has an anti-metastatic function in other types of cancer cells. We further verified that miR-612 targets AKT/ERK signaling pathways and exerts its suppressive role on BC cells through apoptosis.
High mortality rates are related to the metastasis and invasion of the cells in patients with various cancers [19, 20]. The wound-healing approach was used in this regard to assess the effects of miR-612 on the migration of BC cells. As previously mentioned, our study designated that the invasion and migration of miR-612-transfected BC cells substantially decreased compared to the control group. To further investigate the effects of miR-612 on the inhibition of migratory ability and invasion of BC cells, we evaluated the MMP3 and ROCK1 gene expression in miR-612-transfected BC cells. Previous observations revealed that MMP3 and ROCK1 played a pivotal function in cell proliferation, invasion, and metastasis of various cancer cells [21, 22]. Moreover, the MMP3 and ROCK1 genes are overexpressed in approximately all human cancers and considered as potential targets for cancer therapy . The obtained data indicated that miR-612 significantly reduced the expression of MMP3 and ROCK1 in BC cells and thus inhibited the invasion and migration of BC cells.
To examine the effects of miR-612 in BC cell inhibition, the cell cycle arrest was assessed in miR-612-transfected cancer cells. Sub-G1 DNA content in miR-612-transfected cells was assessed by PI staining due to involvement of the G1/G0 cell cycle arrest in apoptosis. Our findings demonstrated that the miR-612 considerably enhanced the number of cells in the phase G0/G1 and blocked the transition in miR-612 transfected cancer cells from phase G1 to S. This indicates the effects of miR-612 on cell cycle arrest and inhibition of BC cell proliferation, which may correlate with apoptosis. To further investigate the functions of miR-612 on the cell cycle arrest in BC cells, we evaluated the cyclin D and PTEN gene expression in miR-612-transfected cancer cells. Previous studies showed the crucial role of cyclin D and PTEN in the cell cycle and apoptosis of various cancer cells [24, 25]. Also, the cyclin D and PTEN genes are dysregulated in approximately all human cancers. Thus, they are considered potential targets for cancer therapy . The obtained results indicated that miR-612 significantly suppressed the expression of MMP3 and ROCK1 in BC cells, indicating that miR-612 may inhibit the cell cycle in BC cells.
Apoptosis induction is an important treatment mechanism in different cancer cells. In our study, the apoptosis of miR-612-transfected BC cells was investigated. The results demonstrated that after transfection with miR-612 apoptosis indices of BC cells were increased. Inconsistent with other studies, miR-612 results in the induction of apoptosis in different tumor cells [26, 27]. The family of caspases proteins plays a significant role in initiating and executing apoptosis. The proteins caspase3 and caspase8 are the most frequently involved in apoptosis in different types of tumor cells . Accordingly, we evaluated the caspase3 and caspase8 gene expression in miR-612-transfected cancer cells. The obtained results revealed that the miR-612 significantly increased both caspase3 and caspase8 gene expression. This suggests that miR-612 exerts satisfactory anticancer efficacy by increasing pro-apoptotic factors and inducing apoptosis in tumor cells. Nevertheless, underlying mechanisms of apoptosis induction by miR-612 have not been revealed. Following the transfection of miR-612, the mRNA expression of AKT and Bcl2 as anti-apoptotic genes was reduced. Additionally, Bax expression as the pro-apoptotic gene was augmented, indicating increased apoptosis.
Also, we assessed the protein levels involved in the AKT/ERK signaling cascade to further investigate whether miR-612 induces apoptosis in the BC cells. Early studies suggested that miR-612 could suppress the potential of hepatocellular carcinoma carcinogenesis and metastasis by targeting AKT2 [13, 29]. We showed that miR-612 inhibited BC cell proliferation and migration by inhibiting the expression of the AKT/ERK signaling pathway. AKT is a serine/threonine kinase that regulates cellular functions including cell migration, proliferation, metabolism, and apoptosis [30, 31]. The AKT/ERK signaling regulates the expression of associated proteins including Bax, Bcl2, and cyclin D1, in the cell cycle and cell apoptosis [30, 31]. The pathways PI3K/AKT and MAPK/ERK are both important cascades of intracellular signaling, regulating cell growth, proliferation, invasion, survival, mobility, and apoptosis [32, 33]. It was observed in the current study that miR-612 strongly decreased the activation of the AKT/ERK signaling cascade in BC cells. Therefore, we suggest that miR-612 induces apoptosis and suppresses proliferation and migration of BC cells by suppressing the AKT/ERK signaling cascade.