Evaluation of the potential diagnostic role of the Lnc-MIAT, miR-29a-3p, and FOXO3a ceRNA networks as non-invasive circulatory biomarkers in ductal carcinoma breast cancer

doi:10.21203/rs.3.rs-1437285/v1

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Evaluation of the potential diagnostic role of the Lnc-MIAT, miR-29a-3p, and FOXO3a ceRNA networks as non-invasive circulatory biomarkers in ductal carcinoma breast cancer

https://doi.org/10.21203/rs.3.rs-1437285/v1

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Over the last few decades, tremendous progress has been achieved in the early detection and treatment of breast cancer. However, the prognosis remains unsatisfactory, and the underlying processes of carcinogenesis are still unclear. The purpose of this research was to find out the relationship between MIAT, FOXO3a, and miRNA29a-3p and evaluated the expression levels of them in breast cancer tissue compared to whole blood and their potential as a noninvasive biomarker in whole blood in breast cancer. Whole blood and breast cancer tissue are taken from patients before radiotherapy and chemotherapy. Total RNA was extracted from breast cancer tissue and whole blood to synthesize complementary DNA (cDNA). The expression of MIAT, FOXO3a and miRNA29a-3p was analyzed by the RT-qPCR method and the sensitivity and specificity of them were determined by the receiver operating characteristic (ROC) curve. Bioinformatics analysis was used to understand the connections between MIAT, FOXO3a, and miRNA29a-3p in human breast cancer in order to develop a ceRNA (competitive endogenous RNA) network. We identified that in ductal carcinoma breast cancer tissue and whole blood, MIAT and FOXO3a were more highly expressed, while miRNA29a-3p was lower compared with those in non-tumor samples. There was a positive correlation between the expression levels of MIAT, FOXO3a, and miRNA29a-3p in breast cancer tissues and whole blood. Our results also proposed miRNA29a-3p as a common target between MIAT and FOXO3a, and we showed them as a ceRNA network. This is the first study that indicates MIAT, FOXO3a, and miRNA29a-3p as a ceRNA network, and their expression was analyzed in both breast cancer tissue and whole blood. As a preliminary assessment, our findings indicate that combined levels of MIAT, FOXO3a, and miR29a-3p may be considered as potential diagnostic biomarkers for breast cancer.

Breast cancer

non-invasive biomarkers

ceRNA network

MIAT

miRNA29a-3p

FOXO3a

bioinformatics

With an estimated 2.3 million new cases of breast cancer(BC) annually (11.7%), it surpassed lung cancer as the most commonly diagnosed cancer in 2020[1]. Both BC incidence and mortality rates are increasing among Iranian women[2]. Iranian women develop this disease at least a decade sooner than women in developed countries and are usually diagnosed with this disease at the age of 40 to 49[3]. However, BC recurrence rates and drug resistance keep rising, so the treatment and prognosis of BC are not satisfactory. Therefore, further research into the molecular etiology of BC is required to identify novel biomarkers and therapeutic targets.

Apoptosis is critical for maintaining a balance between cell death and cell division; thus, deregulation of this pathway leads to uncontrolled cell proliferation, which is involved in many different diseases, such as cancer[4]. The intrinsic pathway of apoptosis is regulated by various factors, such as the Bcl-2 family and the PI3K/AKT pathway[5, 6]. Numerous studies have shown that the PI3K/AKT pathway is involved in the progression of a variety of tumors through regulating cellular growth and blocking apoptosis[7]. It has been reported that AKT interferes with cell death pathways through the phosphorylation of FOXO3a[8, 9]. FOXO3a, a transcription factor of the Forkhead box O (FOXO) family, possesses a vital role in regulating several cellular processes, such as proliferation, apoptosis, DNA damage, and cell cycle progression[10]. Notably, in the PI3K-AKT pathway, activation of FOXO3a induces apoptosis, cell-cycle arrest, and stress resistance in most tissues, while FOXO3a inactivation triggers cell survival, proliferation, and stress sensitivity[11]. In BC, the deregulation of the PI3K-AKT pathway and its increased activity are associated with reduced diagnosis in patients[12].

Long non-coding RNAs (lnc-RNAs) are a sort of non-coding RNA (ncRNA) that are, described as transcripts with a length of more than 200 nucleotides[13]. Recent research has demonstrated that lncRNAs are critical regulators of a variety of biological processes, including innate immunological responses, genetic expression regulation, post-transcriptional processes, proliferation, invasion, metastasis, and angiogenesis of cancer cells[14]. Accordingly, there is significant evidence suggesting lncRNAs function as competing endogenous RNAs (ceRNAs) to restrict microRNA expression or activity. Thus, lncRNA has the potential to be a sensitive cancer diagnostic biomarker[15]. Myocardial infarction associated transcript (MIAT) is involved in the control of cancer cell apoptosis, cell cycle regulation, migration, and invasion[16]. In breast cancer cells, knocking down MIAT inhibited cell growth and promoted apoptosis. It has been reported that, in a xenograft model, the inhibition of breast tumors and decreased MIAT expression are associated with reduced invasion and migration of cancer cells, thus indicating MIAT acts as an oncogene[17, 18].

MicroRNAs (miRNAs), which are classified as non-coding mRNAs with approximately 22 nucleotides in length. Recently, a growing body of research has established that miRNAs control gene expression at the post-transcriptional level[19]. Therefore, changing microRNA expression levels are involved in the onset and progression of a variety of diseases, such as cancer[20]. miRNA-29a-3p has a critical role in different biological processes such as proliferation, apoptosis, and cell cycle regulation. It has been reported that miRNA-29a-3p has a significant impact on cancer development by acting as tumor suppressors[21].

Additionally, miRNA-29a-3p expression levels have been discovered to be abnormally low in a variety of human malignancies, including papillary thyroid carcinoma (PTC), hepatocellular carcinoma, and gastric cancer[22]. Previous research found that miRNA-29a-3p is involved in the progression of BC. Moreover, circRNA ACAP2 (circACAP2) increases BC metastasis and proliferation via sponging miRNA-29a-3p[23].

The purpose of the study was to evaluate the expression levels of MIAT, FOXO3a, and miRNA-29a-3p as a ceRNA network in the BC tissues compared with the whole blood of BC patients, investigate their relationship with the clinical features of the tumor, and examine their potential as noninvasive biomarkers in BC.

2.1. Bioinformatics Analysis

Protein-protein interactions analysis

The STRING server (https://string-db.org/) is used to investigate protein-protein interaction networks. We searched for FOXO3a in the STRING database and found the top 10 proteins that are related to FOXO3a. Then we sorted out these connections based on Text mining, Experiments and, Databases.

2.1.1. Analysis of STRING enrichment

The Gene Ontology (GO) is divided into three categories: biological process (BP), molecular function (MF), and cellular component (CC). Along with GO, the Kyoto Encyclopedia of Genes and Genomes (KEGG) is a valuable resource for studying biological pathways. Thus, we utilized the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the databases to investigate functions and pathways in order to determine their biological meaning.

In this study, after finding the protein-protein interaction of FOXO3a, GO enrichment and KEEG pathway related to FOXO3a protein-protein network from the STRING database were downloaded. They were categorized based on adjusted P value, and the top ten were chosen. Enrichment analysis of FOXO3a was visualized in R 4.0.5 software (https://www.r-project.org/) based on the ggplot2 package (https://cran.r-project.org/web/packages/ggplot2/index.html.).

2.1.2. The interaction between MIAT, FOXO3a, and miRNA29a-3p

The interaction between MIAT, FOXO3a and miRNA29a-3p was determined using TargetScan (http://www.targetscan.org/)and the Starbase database (https://starbase.sysu.edu.cn/index.php .this).

2.1.3. Construction of the ceRNA Network

We predicted the miRNAs that interacted with MIAT and FOXO3a by using the miRNet (https://www.mirnet.ca/)and the miRDB (http://mirdb.org/) databases, respectively. Using miRNet, 46 miRNAs were found for MIAT and by using miRDB, 90 miRNA based on target score were found for FOXO3a.

We combined MIAT-miRNAs and FOXO3a-miRNAs and applied them to Cytoscape software (v3.0.9) (https://cytoscape.org/) then the ceRNA network was constructed based on the results.

2.3. Patients and sample collection

A total of 120 samples were taken from the Imam Khomeini Hospital (Tehran, Iran) among which 70 samples were collected from breast cancer patients (35 blood samples,35 tissue samples), and 50 cases were collected from healthy women (25 blood samples,25 normal breast tissue). The mean age of the patients was 47 ± 3.1 year. All patients had neither chemotherapy nor radiotherapy treatment before surgery. The healthy individuals with no family history of any cancers and diseases, and no history of alcohol consumption were selected. Also, their age (mean age 45 ± 5.4 year) and gender were consistent with the patient samples. The pathological features of patients were indicated in Table 1.

Table 1

Characteristics of the cancer patients and specimens used in this study.
Variable	Total (%)
Age < 40 ≥ 40	18 (51.4%) 17 (48.5%)
Tumor size < 2 cm ≥ 2 cm	9 (25.7%) 26 (74.2)
TNM stage Ⅰ-Ⅱ Ⅲ-IV	25 (71.4%) 10 (28.57%)
ER status Negative Positive	11 (31.4%) 24 (68.5%)
PR status Negative Positive	14 (40%) 21 (60%)
Her2 status Negative Positive	8 (22.8%) 27 (77.1%)
Lymph node Free Involved	13 (37.1%) 22 (62.8%)
Histology Ductal	35

2.4. RNA extraction

Total RNA was isolated from whole blood samples of BC patients and healthy controls according to the manufacturer instructions, using Trizol (Yekta Tajhiz Azma, Iran). The xylene-ethanol technique was used to eliminated paraffin from FFPE tissue, and afterward, overall RNA was extracted using Trizol Reagent (Yekta Tajhiz Azma, Iran) according to the manufacturer’s instructions. RNA quantification and the 260/280 nm ratio was evaluated by NanoDrop NP80 (Implen, Germany). Subsequently, total RNA was treated by RNase-free DNase (SinnaClon, Iran) to eliminated genomic DNA.

2.5. RT- qPCR

High-Capacity cDNA revers-transcription kit (Smobio, Taiwan) was used for cDNA synthesis. Specific primers were designed for MIAT, GAPDH using oligo primer analysis software (version 0.7.0), a specific primer for FOXO3a was used from primer bank Harvard (Gen Bank Accession: NM_001455); and primers of miRNA29a-3p and, U6 are taken from (22). The primer sequences are shown in Table 2.

Table 2

Sequences of primers using for qPCR.
Primer Sequence
MIAT-F 5-CGGAAGCTAGAGGGCGTTA-3 MIAT-R 5-TGCGGGAACCCTGTGAGC-3
FOXO3a-F 5-CGGACAAACGGCTCACTCT-3 FOXO3a-R 5-GGACCCGCATGAATCGACTAT-3
GAPDH-F 5-ATGAGAAGTATGACAACAGCCTC-3 GAPDH-R 5- CATGAGTCCTTCCACGATACC-3
miRNA29a-3p –F 5-GCGTAGCACCATCTGAAAT-3 miRNA29a-3p –R 5- CAGTGCGTGTCGTGGAGT − 3
U6-F 5-CGCAAGGATGACACG-3 U6-R 5-GAGCAGGCTGGAGAA-3

The relative expression was carried out by SYBR Green RealQ Plus 2x Master Mix Green (Ampliqon, Denmark) using the Applied Biosystems StepOnePlus™ (USA). PCR reactions of FOXO3a, MIAT and GAPDH were performed by applying the following thermal protocol: 95°C for 10 min, followed by 40 cycles of amplification (95°C for 15 s, 60°C for 45 s and 60°C for 60 s). PCR reactions of miRNA29a-3p and U6 were incubated at 95°C for 30 s, followed by 40 cycles of 95°C for 10 s, 60°C for 15 s and finally 72°C for 30 sec. The specificity of our RT-qPCR amplification was validated by melting curves analyses. GAPDH and U6 levels were used as reference genes since earlier research on GAPDH and U6 expression levels demonstrated consistent expression in multiple organs, good stability, and little fluctuation in circulation. Thus, our data were normalized by GAPDH and U6. The PCR products were confirmed on 1.5% agarose gel.

2.6. Statistical analysis

All reactions were run in duplicates. The amplification efficiency was analyzed using LinReg PCR software (version 0.1.0). The 2^−ΔΔCt method was used to calculate the relative level of gene expression. The analytical distinction between the groups was evaluated by the one-way ANOVA and independent sample T-test, which was executed by the Graph Pad 8.0.2 software (GraphPad Software Inc., San Diego, CA, USA), and p value < 0.05 was considered statistically notable. Indeed, in order to evaluate the diagnostic ability of MIAT, FOXO3a and miRNA29a-3p and in combination receiver operating characteristic (ROC) curve and area under the curve (AUC) were used. The appropriate cut-off values for MIAT, FOXO3a, and miRNA29a-3p were determined, and based on these cut-off points, the sensitivity and specificity were evaluated.

3.1. Bioinformatics Analysis

3.1.1. Protein-protein interactions analysis

The interaction analysis revealed that FOXO3a protein-protein network is related to NAD-dependent protein deacetylase sirtuin-1(SIRT1), Cyclin-dependent kinase 2 (CDK2), Catenin beta-1 (CTNNB1), NAD-dependent protein deacetylase sirtuin-3(SIRT3), 14-3-3 protein zeta/delta (YWHAZ), RAC-alpha serine/threonine-protein kinase (AKT1), E3 ubiquitin-protein ligase Mdm2 (MDM2), Cellular tumor antigen p53 (TP53), Histone acetyltransferase p300 (EP300) and proto-oncogene protein(MYC) (Fig. 1a). These genes that are involved in protein-protein networks have an important role in the regulation of the apoptotic signaling pathway.

Enrichment analysis based on protein-protein interactions that were constructed in the string database indicated that these genes that are part of protein-protein network in terms of biological process in regulation of response to DNA damage stimulus, apoptotic signaling pathway, cellular senescence and according to molecular function (MF) in disorder domain specific binding, androgen receptor binding, DNA-binding transcription factor binding are involved. Moreover, cellular component showed that they are in the cytosol, nucleoplasm, and protein-containing complex. The KEEG pathway has shown the role of theme in various cancer such as BC, gastric cancer, and non-small cell lung cancer (Fig. 1b).

3.1.2. miR29a-3p has a common binding site with MIAT and FOXO3a

Bioinformatics investigation revealed that FOXO3a (Fig. 2a) and MIAT (Fig. 2b) shared a common miR-29a-3p binding site. Moreover, both MIAT and FOXO3a had a conserved miR-29a-3p binding sites on their 3′-UTRs (Fig. 2c).

3.1.3. The Construction of MIAT and FOXO3a ceRNA Network

LncRNA can decrease the suppression of mRNA expression by competitively binding to miRNA (23). According to this theory, negative connections exist between miRNAs and lncRNAs or mRNAs, whereas positive correlations exist between mRNAs and lncRNAs.

MIAT and FOXO3a in this study were subjected to further bioinformatics analyses to identify the most commonly predicted target miRNAs. The miRNet and miRDB database were used to predict target miRNAs of MIAT and FOXO3a respectively. The combination of miRNAs according to 1 MIAT node, 1 gene FOXO3a node, 136 miRNA nodes, and 136 edges, which were visualized using Cytoscape. So, as it was demonstrated in Fig. 3, miRNA29a-3p is only common target between MIAT and FOXO3a. This finding showed that the MIAT-miRNA29a-FOXO3a network acts as a sponge based on competitive endogenous RNA (ceRNA). According to this theory, MIAT can act as ceRNA network to regulate FOXO3a expression by sponging miR-29a-3p(Fig. 3). However, further research is needed in the future.

3.2. Differential expression of MIAT, FOXO3a and miRNA29a-3p in patients’ whole blood and healthy blood

Our studies showed that the expression levels of MIAT and FOXO3a are remarkably higher in the whole blood of BC patients compared with healthy individuals (p < 0.04 and p < 0.02, respectively). In contrast, the expression level of miRNA29a-3p (p < 0.001) was substantially lower in the patient group compared to the control groups (p < 0.001) (Fig. 4).

3.3. Differential expression of MIAT, FOXO3a and miRNA29a-3p in ductal carcinoma BC tissue and normal breast tissue

Our outcomes revealed that the total mean expression levels of MIAT and FOXO3a were dramatically much higher in BC tissue than normal breast tissue (p < 0.03, p < 0.006 respectively). In comparison to the control groups, miRNA29a-3p expression levels were significantly lower in the patient group (p < 0.05) (Fig. 5).

3.4. Differential expression of MIAT, FOXO3a and miRNA29a-3p based on clinical features of the tumor in ductal carcinoma BC tissue and whole blood

The result of this study indicated that overexpression of MIAT was 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). There was no significant difference in ER, Her2, PR status in BC tissue and whole blood. On the other hand, upregulation of FOXO3a was positively associated with ER-positive (p < 0.02), lymph node involvement (p < 0.01) and TNM stage (p < 0.004) in BC tissue. Moreover, there was a significant difference in the upregulation of FOXO3a and ER -positive (p < 0.04), lymph node involvement (p < 0.01) and TNM stage (p < 0.007) in whole blood, but there was no significant difference in Her2, PR status and RR-negative in BC tissue and whole blood. In contrast, low expression of miRNA29a-3p was clearly associated with ER-positive in both BC tissue (p < 0.006,) and whole blood (p < 0.004) but, significant difference has not been seen between downregulation of miRNA29a-3p and other features of the tumor.

3.5. Assessment of the diagnostic performance of MIAT, FOXO3a, and miRNA29a-3p in whole blood and ductal carcinoma BC tissue

MIAT, FOXO3a and miRNA29a-3p were differently expressed in the whole blood of breast cancer and healthy individuals, hence, ROC curve was applied to predict the potential of these genes in differentiating BC from healthy individuals. MIAT at the cut off 0.8 with a sensitivity of 78%, a specificity of 100% and AUC values 0830 (Fig. 6a), FOXO3a at the cut off 0.9 with a sensitivity of 90%, a specificity of 100% and AUC values 0.900 (Fig. 6b) and miRNA29a-3p at the cut off 0.8 with a sensitivity of 100%, a specificity of 80% and AUC values 0.910(Fig. 6c) detect BC from healthy women respectively.

Additionally, ROC curve was used to assess potential of MIAT, FOXO3a and miRNA29a-3p to distinguish between BC tissue and normal samples. MIAT at the cut off 0.6 with a sensitivity of 88%, a specificity of 80% and AUC values 0.880 (Fig. 6e), FOXO3a at the cut of 0.8 with a sensitivity of 75%, a specificity of 100% and AUC values 0.853(Fig. 6f) and miRNA29a-3p at the cut of 0.8 with a sensitivity of 80%, a specificity of 100% and AUC values 0.940(Fig. 6g) distinguish BC from healthy individuals respectively.

Furthermore, the diagnostic accuracy of the combination of MIAT, FOXO3a and miRNA29a-3p in whole blood and BC tissue was performed. In the whole blood the combination of MIAT, FOXO3a and miRNA29a-3p diagnose BC from healthy women at the cut of 0.9 with a sensitivity of 95%, a specificity of 90% and AUC values 0.990(Fig. 6d) and in BC tissue they detect BC from normal samples at the cut of 0.9 with a sensitivity of 90%, a specificity of 100% and AUC values 0.100(Fig. 6h).

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[24]. Apoptosis includes a large number of signaling pathways, which is considered a precise regulatory mechanism[25]. 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[26].

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[27]. Ying Song et al. reported that upregulation of FOXO3a substantially inhibited BC cell migration and invasion in vitro[28]. 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[31]. 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[32]. ER has a critical role in growth, proliferation, and differentiation in BC[33]. There is abundant evidence to show crosstalk between FOXO3a and ER signaling pathways[34]. 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[30]. J. Chen et al. observed that upregulation of FOXO3a was correlated with increased AKT expression and lymph node metastases[35]. 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[36]. 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[18]. 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[37]. A recent study showed that the expression level of MIAT is increased in ER-positive BC tissue and cell lines[38]. It has been reported that MIAT is significantly expressed in stage I and stage II breast tumors[39]. 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[40]. 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[41]. 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[42].

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[43]. 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[44].Pei YF et al. proposed that higher miRNA-29a-3p expression increased cell proliferation, while decreased miRNA-29a-3p expression suppressed cell growth[45]. 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[46]. 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[47]. 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[48]. Increasing evidence indicates that lncRNA functions as a ceRNA, inhibiting the expression or activity of miRNA[49]. 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[51]. 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[52].

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[53]. 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.

Ethical approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Islamic Azad Tehran Medical Sciences University (code: IR.IAU.PS.REC.1398.317 dated January 21, 2020).

Consent to participate

Informed consent was obtained from all individual participants included in the study.

Consent to publish

The authors affirm that written informed consent was obtained from all individual participants to publish this paper.

Authors’ Contributions

HM designed, supervised and approved the final manuscript. SHR, contributed to all experimental work, data and statistical analysis, interpretation of data and wrote the manuscript draft. RBA Was responsible for overall supervision. All authors read and approved the final manuscript.

Competing interests

The authors have no competing interests to declare that are relevant to the content of this article.

Availability of data and material

The datasets generated or analyzed during the current study are available from the corresponding author on reasonable request.

Funding

No funding was received for conducting this study.

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No competing interests reported.

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Evaluation of the potential diagnostic role of the Lnc-MIAT, miR-29a-3p, and FOXO3a ceRNA networks as non-invasive circulatory biomarkers in ductal carcinoma breast cancer

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Bioinformatics Analysis

2.1.1. Analysis of STRING enrichment

2.1.2. The interaction between MIAT, FOXO3a, and miRNA29a-3p

2.1.3. Construction of the ceRNA Network

2.3. Patients and sample collection

2.4. RNA extraction

2.5. RT- qPCR

2.6. Statistical analysis

3. Results

3.1. Bioinformatics Analysis

3.1.1. Protein-protein interactions analysis

3.1.2. miR29a-3p has a common binding site with MIAT and FOXO3a

3.1.3. The Construction of MIAT and FOXO3a ceRNA Network

3.2. Differential expression of MIAT, FOXO3a and miRNA29a-3p in patients’ whole blood and healthy blood

3.3. Differential expression of MIAT, FOXO3a and miRNA29a-3p in ductal carcinoma BC tissue and normal breast tissue

3.4. Differential expression of MIAT, FOXO3a and miRNA29a-3p based on clinical features of the tumor in ductal carcinoma BC tissue and whole blood

3.5. Assessment of the diagnostic performance of MIAT, FOXO3a, and miRNA29a-3p in whole blood and ductal carcinoma BC tissue

4. Discussion

Declarations

Ethical approval

Consent to participate

Consent to publish

Authors’ Contributions

Competing interests

Availability of data and material

Funding

References

Additional Declarations

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