Over the past few years, microRNA dysfunctions have been listed as a major factor in human cancer incidence. Emerging evidence indicates that microRNAs play a critical role in the emergence and progression of tumors. Moreover, these molecules are able to modulate the expression level of different oncogenes and tumor suppressor genes, which are the most relevant markers of cancer biology [17].
Increasing evidence has obviously demonstrated that miR-451 and miR-34 (separately) are notably dysregulated in various human malignancies and determine an essential role in tumorigenesis and tumor development, which mainly act as tumor suppressors and can modulate cell proliferation, motility, and migration by a variety of specific targets like c‐Myc and MMP-9 [18–20]. In this connection, emerging documents reported that the levels of miR‐451 and miR-34 are reduced in human BC specimens, which was related to the poor prognosis and lymph node metastases [18, 21, 22].
In the present study, firstly, we showed that the expression levels of miR-451a-5p and miR-34a-5p are low in BC cell lines, particularly in the MBA-MD-231, which was selected for further analyses. These findings demonstrated that both microRNAs act as the tumor suppressors in BC, which agreed with the previous studies [18, 20, 22].
In the last decade, miRNAs have been suggested as specific potential predictive, diagnostic, and prognostic strategies in a variety of diseases like human cancers. Importantly, those also are attended as therapeutic tools in cancer treatment by promoting or suppressing special molecular pathways. Of interest, it has been proposed that silencing an oncogenic miRNA or restoring a tumor-suppressive miRNA might represent a beneficial antitumor therapy [23]. Therefore, miRNA-replacement therapy can be used as a treatment strategy in cancers, which was used for the first time in 2009 [24].
Thus, the present study aimed to evaluate the restoration effects of miR-451a-5p and miR-34a-5p, individually and combined, on the biology of breast cancer in vitro, conducted by transfection miR-451a and miR-34a mimics into MDA-MB-231 cells.
Here, MTT assay, flow-cytometric analysis, DAPI staining, and expression levels of caspase-3 and caspase-8 (by qRT-PCR) were used for the detection of apoptosis upon restoration of miR-451a and miR-34a and their combination in BC cells. After transfections, the viability of the cancer cells notably decreased. The apoptotic rate was increased and reached 74.5%, apoptotic cells strongly increased in treatment groups by DAPI staining, and the expression levels of caspase-3/8 were remarkably increased. These results showed that apoptosis was strongly enhanced after miR-451a and miR-34a transfections, particularly in the combination group. Caspases-3 and caspase-8 are members of aspartate-specific cysteine proteases and contribute to the extrinsic programmed cell death pathway following external stimulation of the death receptors as executioner and initiator caspases, respectively. In this study, expression levels of both caspases were significantly increased after miRNAs restoration. Our findings are close to those of others, which strongly indicated the anti-apoptotic function of miR-34a and miR-451a in BC. In this line, it was previously reported that overexpression of miR-184, miR-216a, and miR-145 that act as tumor suppressors in breast cancer cells can induce apoptosis via activation of caspase-3/8 and subsequently inhibit proliferation, invasion, and adhesion of cancer cells [25–27].
In the present study, the results of proliferation and migration analyses, including wound healing assay and expression levels of MMP9, vimentin, and ROCK genes (by q-RT-PCR), demonstrated that the replacements of miR-451a, miR-34a, and miR-451a-34a-combination notably inhibited the migration ability of the cancer cells, especially in individual groups. MMP-9, a member of zinc-dependent endopeptidases, triggers cancer development and progression in most cases [28] associated with a higher expression level in BC cells, particularly in triple-negative and HER2-positive breast cancers [29, 30], which was related to tumor metastasis and staging [29]. Our data is consistent with some of these researches. Indeed, the present results showed downregulation of MMP-9 after miRNAs transfection, particularly by miR-451. Vimentin has also been demonstrated as an indicator for pre-metastatic cancer cells undergoing epithelial to mesenchymal transition (EMT). Thus, as expected, its overexpression is related to poor outcomes in patients with solid cancer [31]. In this way, several studies revealed the contribution of miRNAs to the EMT process in BC cells through vimentin expression [32, 33]. The present findings are also similar to the recent studies. As more details, downregulation of vimentin was found after miRNAs overexpression, particularly by miR-34a transfection. ROCKs are known as the substantial manager of focal adhesion formation, cancer cell motility, and invasion [34]. Growing evidence indicated the downregulation of ROCK1 by miR-193a and miR-340 in breast cancer [32, 35]. The remarkable higher ROCKs protein levels were reported in breast cancer patients, and it seems that it may associate with tumor grade progression and poor overall survival. Previously, it was reported that miR-340 and miR-148b could modulate cell migration, invasion, metastases, and cancer progression in BC by targeting ROCK [35, 36]. Here, as shown by qRT-PCR, the expression level of ROCK was considerably decreased after miRNAs overexpression, particularly in the miR-451 group.
In the current study, overexpression of miR-451a and miR-34a considerably arrested the cell cycle in the sub-G1-phase, particularly in the combination group. As more details, 64.8%, 58.8%, and 69.6% of the cells were in sub G1 phase in miR-451, miR-34, and combination group, respectively. Indeed, upregulation of miR-451 and miR-34 prevented mitotic entry by inducing sub G1 arrest and eliminated cells with impaired DNA. Besides, in this study, the expression level of c-Myc as a major cell cycle promoter oncogene in human breast cancer cells was reduced obviously. Indeed, c-Myc is a transcription factor that presents in the cell nucleus, which is frequently dysregulated and overexpressed in many human cancers [37]. A previous study investigated the c-Myc as a specific target of miR-451, which can regulate the proliferation and migration of cancer cells [19]. These data documented that overexpression of miR-451a and miR-34a, individually and combined, inhibit BC cell proliferation and induce cell cycle arrest in the sub-G1-phase.
Based on the present western blot analyses, miR-34a-5p transfection did no significant alteration in β-catenin protein expression levels. But, miR-451a-5p individually and combined with miR-34a significantly downregulated the β-catenin protein levels. It was previously revealed that overexpression of miR-451 could strongly inhibit the expression of some mesenchymal markers like β‐catenin [38]. β- Catenin is a multifunctional protein that functions both as a transcriptional co-modulator and an adaptor protein for intracellular adhesion. Of note, Wnt is the chief regulator of β-catenin. Following Wnt stimulation or genetic modifications of Wnt components, β-catenin deposits in the cytoplasm and then translocates into the nucleus. Next, it can trigger the transcription of some target genes such as c-Myc [39]. A similar study reported that a high expression level of β-catenin (in cytoplasm or nucleus) commonly promotes tumorigenic features and increases cancer cell proliferation and viability [40]. Wnt/β-catenin signaling has been broadly implicated in human cancers, which is tightly linked with tumor progression, malignancy, poor prognosis, and short survival [41]. Recently, it was demonstrated that miR‐451 upregulation could negatively modulate the Wnt/β‐catenin pathway via the c‐Myc of β‐catenin [22]. Our results are in agreement with the previous studies.
In the current study, western blot analyses also showed that restoration of miR-451a/34a increased the protein expression levels of ERK and P-ERK (phosphorylated-ERK) in BC cells. ERK (extracellular signal-regulated kinase 1/2) belongs to the MAPK family (the mitogen-activated protein kinase), which functions in signaling pathways and transmits extracellular signals to intracellular receptors. ERK cascade plays an essential role in several cellular processes like cell proliferation, adhesion, differentiation, migration, and survival [42]. Increased ERK expression level has been previously determined in various human cancers such as BC [43]. On the other hand, the expression of MMP9 depends on the phosphorylation of ERK and the presence of P-ERK. Our data interestingly indicated that miR-451a/34a reduces ERK and P-ERK expression levels and subsequently leads to repression in cell survival, proliferation, and migratory ability. Restoration of miR-451a/miR-34a reduced the activities of ERK/P-ERK and approved that the miR-451a and miR-34a have a notable role in the RAS-RAF-ERK and associated signaling pathways. Indeed, this finding confirms the results of the MTT test, flow cytometry assay (to find apoptotic rate and cell cycle arrest), wound healing assay, and qRT-PCR analyses.