The existing diagnosis approach cannot detect cancer early and it influences the life quality of patients. As a result, it is critical to find a biomarker that would be accessible, cost-effective, and sensitive enough to detect and monitor BC patients. The purpose of this research was to determine the interaction between MIAT, FOXO3a, and miRNA29a-3p and compare the expression levels of them in BC tissue with whole blood to see if they could be useful biomarkers for ductal carcinoma BC diagnosis and management in clinical practice. Our study revealed the overexpression of MIAT and FOXO3a, and the downregulation of miRNA 29a-3p in the tissues and whole blood of Iranian women with BC ductal carcinoma (Fig. 4–5). The AUC values for MIAT, FOXO3a, and miRNA29a-3p indicated that they are effective candidates with a high degree of specificity and sensitivity for the diagnosis of ductal carcinoma BC.
Moreover, MIAT-miRNA29a-3p-FOXO3a have been seen as the ceRNA networks in BC. One of the main strengths of the present study is the construction of the ceRNA network using bioinformatics analysis based on MIAT-targeting and FOXO3a-targeting. According to Fig. 3, miRNA29a-3p was considered the only common target between FOXO3a and MIAT. The MIAT/miRNA-29a-3p/FOXO3a ceRNA network has been described for the first time, which may help us identify a novel ceRNA network involved in the regulation of BC.
The apoptotic pathway is critical for tumor growth and metastasis at all stages. Apoptosis is a biological process that contributes significantly to the growth and survival of multicellular organisms by eliminating damaged, old, or autoimmune cells through a regulated cell death mechanism. Apoptosis includes a large number of signaling pathways, which is considered a precise regulatory mechanism. Therefore, any sort of alternation in these pathways leads to tumorigenesis, metastasis, and resistance to anticancer drugs. Thus, cell cycle control mechanisms have emerged as a potential therapeutic strategy.
In this study, the STRING database and enrichment analysis based on biological process and KEEG pathway indicated that FOXO3a protein- protein network has a critical role in the regulation of the apoptosis signaling pathway and has both positive and negative effects on the regulation of the intrinsic apoptosis pathway (Fig. 1a-b). Among them, MYC, TP53, CDK2, AKT, and MDM2 are important regulators in cell cycle progress and the apoptosis pathway.
It has been reported that FOXO3a acts as a metastasis suppressor because it increases the expression level of E-cadherin and downregulates EMT transcription factors. This resulted in the reversal of the invasive behavior of breast cancer cells. Ying Song et al. reported that upregulation of FOXO3a substantially inhibited BC cell migration and invasion in vitro. In the present study, we have shown significant up-regulation of FOXO3a in breast cancer tissues and whole blood compared to normal groups. In accordance with our results, the overexpression of FOXO3a in breast tumor tissues and cell lines has been shown in some studies[29, 30]. It also has been shown that upregulation of FOXO3a promotes growth of cancer cells and tumor progression. However, recent studies have suggested that the low expression of FOXO3a induces epithelial-to-mesenchymal transition (EMT) and subsequently promotes cancer cell invasion and proliferation, which are associated with breast cancer development and poor response to therapy. ER has a critical role in growth, proliferation, and differentiation in BC. There is abundant evidence to show crosstalk between FOXO3a and ER signaling pathways. Jiang et al. established a clear correlation between FOXO3a expression and ER-positive in human breast tumors and recognized FOXO3a as a promising prognostic marker. They proposed that a high level of FOXO3a expression was shown to be strongly associated with long-term survival in ER-positive cell lines. Moreover, they confirmed that FOXO3a expression was associated with lymph node involvement and TNM stage. J. Chen et al. observed that upregulation of FOXO3a was correlated with increased AKT expression and lymph node metastases. Along with these findings, our analysis showed that, overexpression of FOXO3a was positively associated with ER-positive, lymph node involvement, and TNM stage in BC tissue and whole blood.
MIAT regulates a variety of signaling pathways in cancer. In the study by Yang et al. it was reported that MIAT has been identified as a critical factor in cell invasion, migration, and proliferation through the PI3K/AKT signaling pathway. MIAT dramatically increased PI3K and AKT phosphorylation and stimulated the production of C-MYC and cyclin D1. Recent literature indicated that the expression level of MIAT was higher in BC cells than in normal cell lines, as well as suggesting that MIAT may serve as an oncogene in BC, sharing miRNA-155-5p response element with DUSP7 and promoting BC progression. Alipoor et al. established for the first time that MIAT is implicated in the incidence and progression of breast cancer, presenting it as a potential tumor marker for breast cancer detection and therapy. Their findings have suggested MIAT was up-regulated in BC tissues and cell lines, and our study showed that the expression level of this gene in the whole blood was consistent with its expression in the BC tissues and confirmed the previous data, so it has the potential to be considered as a non-invasive biomarker in the whole blood for BC. They discovered that MIAT expression was considerably greater in high-grade breast ductal carcinoma than in surrounding non-tumor tissues and was associated with clinic pathological characteristics of tumors, such as the human epidermal growth factor receptor 2, the p53 gene, the estrogen receptor (ER), and the progesterone receptor (PR). Their findings indicated that the expression level of MIAT was significantly increased in ER and PR-positive tumor tissues. Moreover, MIAT has recently been found to be overexpressed in P53-negative tumor tissues. Additionally, it was shown that inhibiting MIAT expression resulted in G1-phase arrest and apoptosis in BC cells. These findings suggest that MIAT may act as a cell cycle regulator. Besides, MIAT inhibition prevents breast cancer cell migration and decreases the expression of EMT genes. A recent study showed that the expression level of MIAT is increased in ER-positive BC tissue and cell lines. It has been reported that MIAT is significantly expressed in stage I and stage II breast tumors. In a study by Ting Ye et al. it was shown that higher expression of MIAT was positively related to lymph node status and TNM stage in BC, and they proposed that MIAT serve as a non-invasive biomarker for the diagnosis of BC. The result of this study indicated that the expression level of MIAT was increased in BC tissue and whole blood of patients. Furthermore, overexpression of MIAT was clearly associated with stage I and stage II breast tumors and lymph node involvement in both BC tissue (p < 0.01, p < 0.03 respectively) and whole blood (p < 0.04, p < 0.002 respectively).
A substantial amount of experimental data has demonstrated that miRNAs play a critical role in cancer cell death regulation. Numerous strategies have been developed to either inhibit the expression of oncomiRs or to increase the expression of tumor suppressor miRNAs in an attempt to re-establish miRNA activity in apoptotic pathways. In breast cancer cells, miRNA-29a-3p has been demonstrated to have a tumor suppressor function by interrupting the cell cycle during the G0/G1 phase via the negative regulation of the expression of CDC42.
In the study by Zhi-hua L et al. it was shown that miRNA-29a-3p mimic promoted the proliferation of BC cell lines (MCF-7 and T47D). The inhibition of miRNA-29a-3p was shown to suppress the proliferation of these cell lines. It has been reported that miRNA29a-3p has a negative effect on N-MYC, which leads to upregulation of the mesenchymal phenotype and promotes tumor invasion in BC cells.Pei YF et al. proposed that higher miRNA-29a-3p expression increased cell proliferation, while decreased miRNA-29a-3p expression suppressed cell growth. Wu Z et al. found that in BC cells, the expression level of miRNA29a-3p was decreased. Moreover, miRNA-29a-3p inhibited cells in the G0/G1 phase and restricted tumor development through decreasing the expression of B-MYB. Bin Yan et al. revealed that miRNA29 family members (miR-29a, miR-29b, and miR-29c) increase p53 levels and trigger apoptosis in a p53 pathway. In this research, miRNA-29a-3p was down regulated in BC tissue and whole blood of patients compared to control groups. In assessing the relationship between miRNA29a-3p expression and clinical features of tumor, we found that overexpression of miRNA29a-3p was clearly associated with ER-positive in both BC tissue (p < 0.006,) and whole blood (p < 0.004).
Due to advancements in high-throughput sequencing and novel computing technologies, lncRNA has been recognized as a key molecule in the regulation of gene expression at the post-transcriptional level in recent years. Increasing evidence indicates that lncRNA functions as a ceRNA, inhibiting the expression or activity of miRNA. miRNA has been recognized as a critical regulatory element in the ceRNA network and has a negative impact on regulating RNA gene expression by interacting with the target region of mRNA 3′UTR, causing adenosine acidification, decreasing mRNA stability, and limiting translation[49, 50]. The bioinformatics analysis of the present study suggested that miRNA29a-3p has a common binding site with MIAT and FOXO3a (Fig. 2). Microarray research identified a binding site between miRNA-29a-3p and FOXO3a, showing a similar targeting connection between miRNA-29a-3p and FOXO3a. Growing evidence is emerging to connect miRNA-29a-3p downregulation to FOXO3a overexpression. In another similar study, dual-luciferase demonstrated miRNA-29a-3p has a targeting relationship with FOXO3a in OC (ovarian cancer) and, according to western blot, overexpression of miRNA-29a-3p inhibited the expression of FOXO3a and downregulation of miRNA-29a-3p elevated the expression of FOXO3a. Based on these results, they showed that FOXO3a could be targeted by miRNA-29a-3p.
Additionally, it has been reported that MIAT has complementary base pairing sites with miRNA-29a-3p and it may function as an endogenous miRNA sponge to inhibit the expression of miRNA-29a-3p in gastric cancer. However, further research is needed to determine the exact mechanism of miRNA29a-3p activity in BC. In this study, we showed that the expression levels of MIAT and miRNA29a-3p in whole blood and ductal carcinoma BC tissues have a negative correlation. In fact, overexpression of MIAT is associated with decreased miRNA29a-3p and increased FOXO3a expression. Following these data, it can be concluded that MIAT, miRNA-29a-3p, and FOXO3a levels have a significant relationship with the pathogenesis of ductal carcinoma BC. To confirm the endogenous connection between miRNA-29a-3p, MIAT, and FOXO3a in breast cancer, we propose that future investigations use a dual-luciferase test.
Considering the fact that an ideal biomarker should have high sensitivity and specificity, this study confirmed that MIAT, FOXO3a, and miRNA29a-3p, as well as the combination of them, have shown high sensitivity and specificity in BC tissue and whole blood compared to healthy individuals. Together, these findings indicate that they can be potential biomarkers for BC patients in the whole blood with better sensitivity and specificity.
In summary, it is appealing to demonstrate that MIAT and FOXO3a with high expression and miR-29a-3p, with down expression as a ceRNA network, can be potentially effective biomarkers for the detection of BC in clinical practice. The crosstalk between ncRNAs may provide hope for an accurate diagnosis of BC in the future.