Breast cancer (BC) is the second most common cause of death in women worldwide.¹–³ It arises from uncontrollably altered cell proliferation or function in breast tissue. These alterations cause these cells to become malignant and capable of spreading.⁴,⁵ Biomarkers that aid in monitoring treatment effectiveness may be particularly beneficial.⁶ Regulating genes after transcription is the job of microRNAs, a type of noncoding RNA that is only 22 nucleotides long. Differentiation, cell proliferation, metabolic processes, and cell death are all affected by the ability to impede mRNA translation. Recent studies have shown a connection between abnormal miRNA expression and the onset of many disorders including cancer.⁷,⁸ A member of a similar family of cell surface glycoproteins is a clinically significant tumor marker. Tumor tissue extracts and normal fetal gastrointestinal tract epithelial cells contain this tumor marker, which is useful for identifying colorectal, gastrointestinal, lung, and breast cancers. Lysine is located at the N-terminus of CEA, which is a glycoprotein. CEA consists of one polypeptide chain with 641 amino acids and 45-50% carbohydrates.⁹,¹⁰ Serum CEA levels tend to increase in patients with advanced BC. The clinical use of blood CEA in tracking treatment efficacy in metastatic BC patients, particularly those with bone metastases, has been shown in our research.³,¹¹,¹² Human macrophages and mouse adipocytes release the hormone protein resistin, which has a molecular weight of 12 kDa and is rich in cysteine. It has 108 amino acid peptides and is a progenitor of the resistin-like molecule (RELM) hormone family. In humans, it circulates as a dimer of two 92-amino acid polypeptides.¹³ Resistin enhances epithelial-mesenchymal transition and stemness in BC cells via a signalling pathway involving TLR4, NF-κB, STAT3, and TLR4-specific antibodies and antagonists. These pathways are crucial for metastasis and tumorigenesis.¹⁴ Resistin enhances MCF-7 cell migration and invasion to varying degrees, and its role (MCF-7 cell line) is a key model for studying BC due to its well-characterized estrogen receptor-positive (ER+) status, its ability to form tumors in vivo, and its use in drug resistance and stem cell research. Research has showed that resistin encourages BC cells to invade and migrate¹⁵ in Iraqi women diagnosed with BC. in a study aimed to evaluate the expression of miRNA-425 and miRNA-373 genes as well as the blood levels of CEA and resistin.

This study included 100 womens with BC who attended Al-Amal Hospital in Baghdad, Iraq, for the period from June 2023 to October 2023. The experimental work was carried out at the Institute for Genetic Engineering and Biotechnology Institute for Postgraduate Studies at the University of Baghdad and laboratories of Al-Amal Hospital. The parameters were evaluated using questionnaires and medical records, including disease grade, human epidermal growthfactorreceptor2 (HER2) receptors, age, tumor stage, family history, type, location, estrogen (ER), and progesterone (PR). Women who were 20 years or older and had a history of BC without treatment were included. Individuals with other malignancies, chronic inflammatory illnesses, history of exposure to hormone treatment, radiation, or chemotherapy, and individuals with other cancers were excluded from the study. Overall, 100 healthy women were age-matched with BC patients to rule out their effect on genetic results.

There were different results in this study regarding the presence of luminal B, luminal A, her2+, ER/PR−, and triple negative. Luminal B was seen in 28% of patients, luminal A in 44%, her2+ in 8%, and triple negative in 20%. In 2024, Mohsin and Mohamad (2024) found that 46.67 percent of hormone receptors were positive, 42.22 percent were negative, 62.2 percent were negative for HER2, and 46.67 percent of all molecular subtypes belonged to luminal A and B.¹⁶

The CEA level was 104.05 (261.24) in the control compared to 26.85 (16.47) in the HCs group, which was close to a study which found that metastatic and recurring BCs were associated with significantly higher blood CEA levels in patients.¹⁷ Serum CEA positive rates have been reported in investigations ranging from 36% to 70%. These heightened levels are recognized to be positively correlated with tumor burden, tumor grade, and metastatic site, consequently leading to reduced overall survival (OS) and progression-free survival.¹⁸

The recommendation for serial monitoring of tumor markers is not supported in asymptomatic individuals after BC treatment.¹⁹,²⁰ The commonly used serum tumor markers for BC are CA15-3 and CEA.²¹,²². Therefore, the estimation of serum CEA levels can be considered an adjunctive method for evaluating treatment response, monitoring progress, and obtaining prognostic insights. Nevertheless, the clinical applicability of these markers remains uncertain because of conflicting outcomes.²³,²⁴

In the present study, Serum levels of RETN were significantly higher in BC group in comparison with HCs. Our findings are in agreement with the results of several previous articles.²⁵–²⁷ The study conducted by Hou et al. (2007) collected blood samples from 80 recently diagnosed BC patients who had received histological confirmation, along with 50 age-matched healthy controls.²⁵ The findings indicated that the serum concentrations of resistin were elevated in BC patients compared to healthy controls, with levels of (26.35±5.36) μg/L versus (23.32±4.75)μg/L, showing a statistically significant difference (P=0.000). Dalamaga et al. observed that the average serum resistin levels were notably higher in the cases than in HCs (P<0.001).²⁶ Furthermore, in a study conducted by Assiri et al., a cohort of 82 newly diagnosed BC patients who had histological confirmation and 68 healthy controls matched in terms of age and BMI were included.²⁷ The results revealed significantly elevated levels of resistin in BC patients compared with their respective control counterparts. However, the results of Georgiou et al., were inconsistent with those of the present study as they reported that women with breast carcinoma exhibited resistin levels (6.11±4.49ng/ml), whereas control subjects showed a mean level of 6.14±1.83ng/ml and according to the statistical analysis, there was no notable variation in serum resistin levels between the groups (P=0.064).²⁸

Consistent with the results of the present study, ROC curve analysis showed that resistin had poor diagnostic performance, with a total accuracy level of 72%.²⁶ According to a number of studies, resistin is essential for cancer cell metabolic regulation, angiogenesis, inflammation, proliferation, and metastasis.²⁹. Resistin may serve as a biomarker for BC, indicating an advanced disease stage and inflammatory state, even if its diagnostic performance was poor according to ROC curve study [0.72, 95% CI: 0.64-0.79].²⁶ Research has identified resistin as a possible biomarker for cancer diagnosis and prognosis.²⁵ Researchers have found that resistin levels were higher in BC patients (80 total) than in healthy controls (50 total). Additionally, resistin levels have been shown to be higher in individuals with lymph node metastasis than in those without it.³⁰ Age, menopausal status, blood glucose, body mass index (BMI), and adiponectin levels had no effect on the association between resistin levels and an increased risk of BC. The study conducted by Dalamaga et al. and Assiri et al. found a strong association between tumors and inflammatory markers, tumor size, malignancy grade, stage, and lymph node invasion.²⁶,²⁷ Hou et al. documented that serum concentrations of resistin exhibited notable discrepancies between individuals afflicted with lymph node metastasis and those devoid of such metastasis.²⁵ Also, Dalamaga et al. noted in their study that resistin was significantly correlated with various parameters in BC patients, including cancer stage, tumor dimensions, grade, and lymph node infiltration, while exhibiting no apparent relationship with hormone receptor status.²⁶ In addition, according to Assiri et al. (2015), histological grading, tumor size, lymph node metastasis, and TNM staging were positively correlated with serum resistin levels.²⁷ Our study found a strong negative link between family medical history and a strong positive relationship between RETN, tumor grade, and ER expression. Research suggests that microRNAs play important roles in a variety of cellular processes, and patterns of miRNA expression might be useful indicators for the diagnosis of different cancers and patient outcomes. Additionally, multiple studies have shown that miRNA-373 has oncogenic functions in human cancers by targeting certain genes.¹⁷,²⁴,³¹,³² Nevertheless, the functions of miRNA-373 in BC have been debated by Wei and Wang, who motivated us to examine the role of miRNA-373 in BC.³³

The current study demonstrated a notable increase in the expression levels of miR-373 among patients compared to HCs. This observation aligns with the results reported by Bakr et al., who noted a heightened presence of miRNA-373 in BC patients and those with benign breast lesions, in contrast to reduced levels in the HCs. These results suggest that miRNA-373 could serve as a valuable molecular biomarker for the early detection and diagnosis of BC, facilitating the differentiation between cancerous and non-cancerous instances.³⁴

Interestingly, these findings contradict the observations of Bakr et al., who identified a significant link between the expression level of miRNA-373 and adverse prognostic factors in BC.³⁴ In addition, Raheem et al. found elevated levels of MiRNA-373 in BC pataints (-12.20 ± 1.11). miRNA-373, which is unique to human embryonic stem cells (ESCs), exerts novel oncogenic effects by regulating the growth and formation of tumors in primary human cells containing oncogenic factors such as rat sarcoma (RAS) and wild-type p53.³³ Its distinctive expression patterns and strong relationship with cancer cell invasion and metastasis make it a promising candidate as a potential biomarker for BC, as suggested by findings from studies on cancer cells and tissue samples.³⁵,³⁶ This study examined miR-425 expression levels, which were much higher in patients than in healthy controls. The role of miR-425 in the development of certain cancers was suggested by Zhang et al.¹⁰ The results of this study are in agreement with those of Xiao et al., who used RT-qPCR to show that miR-425 expression was higher in BC tissues than in neighboring tissues and cell lines. In addition, compared to human mammary epithelial cells, BC cell lines exhibit higher expressions of miR-425. To our knowledge, our study is the first to compare miR-425 expression levels in healthy individuals with BC patients in Iraqi women. The significant increase in miR-425 levels observed in our study highlights the promising role of this biomarker as a non-invasive method for detecting early stage.³⁷

While miR-425-5p's involvement in the oncogenesis of other neoplasms has been established, the exact nature of its function in BC remains unknown.¹⁰ This study found that miR-425-5p is greatly upregulated in BC cells and is associated with poor prognosis in BC patients. Zhang et al. (2020) found that miR-425-5p was significantly upregulated in BC cells and predicted a poor prognosis in BC patients.³⁸ BC cell growth was greatly enhanced by an increase in miR-425-5p levels. Following this, researchers found that cyclin-dependent kinases (CDK4), Cyclin D3 (cyclin D3), and miR-425-5p protein levels were all upregulated. Conversely, cell cycle arrest in the G0/G1 phase was the end outcome of miR-425-5p suppression, which led to reduced expression of these genes.¹⁰ From a mechanistic standpoint, silencing miR-425-5p had the opposite effect as overexpression, leading to increased phosphorylation of phosphoinositide 3-kinase- regulatory subunit -p85 (PI3K- p85) and protein kinase B (AKT). Additionally, miR-425-5p binds to the 3'UTR of Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) mRNA, which in turn reduces the production of PTEN in BC cells, both at the mRNA and protein levels. In summary, the results demonstrate that miR-425-5p is involved in BC carcinogenesis via PI3K/AKT pathway activation, implying that it may be a therapeutic target for BC. This study provides important insights into the expression of miR-373, miR-425, and CEA, and serum levels. These findings may be more applicable to the specific patient population and healthcare setting studied, but they lay the groundwork for further research. Larger multicenter studies with more diverse patient populations are essential to confirm and extend these results. Future research should be conducted by increasing the sample size, including more diverse demographic groups, and incorporating multi-center designs. This would enhance the external validity of the findings and provide a clearer picture of how the results can be applied to different settings and populations.

The biomarkers CEA, RETN, miR-373, and miR-425 were identified as promising candidates for enhancing the diagnostic workup, prognosis, and therapy of BC. CEA and RETN are established markers that aid in the early detection and monitoring of tumor progression, whereas miR-373 and miR-425 are involved in critical processes such as tumor metastasis, EMT, and chemoresistance. Their potential to regulate CD44 expression highlights their roles in inhibiting cancer cell invasion and reducing metastasis.