|Year : 2018 | Volume
| Issue : 1 | Page : 81-86
Evaluation of microRNA-155 as a diagnostic serum-based biomarker in patients with breast cancer
Elham Metawae1, Nader Abd-Alhamid2, Salama Abd-Allatif1, Nagwa Abd-Elghaffar3, Eman El-Ghoroury3, Amany Abd-Elrahman3, Ingy Ashmawy3, Zahraa Aboafya3
1 Department of Clinical Pathology, Faculty of Medicine (Boys), Al-Azhar University, Cairo, Egypt
2 Department of Surgery, Faculty of Medicine (Boys), Al-Azhar University, Cairo, Egypt
3 Department of Clinical Pathology, National Research Center, Cairo, Egypt
|Date of Submission||02-Jun-2018|
|Date of Acceptance||26-Aug-2018|
|Date of Web Publication||20-Nov-2018|
Department of Surgery, Faculty of Medicine (Boys), Al-Azhar University, Cairo
Source of Support: None, Conflict of Interest: None
Background Breast cancer (BC) is the most common cancer among Egyptian women, and its early detection is accompanied with decreased mortality rates. MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate gene expression and affect cancer development. The expression level of miRNA-155 is up-regulated in BC and correlates with its invasive properties, offering a great potential as a noninvasive biomarker for BC screening.
Objective The aim of this study is to evaluate the role of miRNA-155 as an early diagnostic serum-based biomarker of BC.
Patients and methods This study was conducted on 30 patients newly diagnosed with BC and associated 20 healthy women as a control group. Serum level of miRNA-155 was measured for all studied cases using real-time quantitative PCR technique.
Results MiRNA-155 expression was up-regulated in serum of patients with BC compared with control group (P<0.001). Serum miRNA-155 expression significantly differed with cancer stage and its expression directly increased with advancing cancer stage. A comparative study of the miRNA-155 expression between control group and positive and negative metastatic lymph nodes (LNs) subgroups demonstrates a highly significant difference. MiRNA-155, in terms of fold change, is highest in positive LNs metastatic subgroup, with a mean of 19.63, followed by 4.29 in negative LNs metastasis subgroup, and lowest in the control group, with a mean of 1.06 (P<0.001).
Conclusion The results validate miRNA-155 as a promising diagnostic and even a prognostic marker in cases of BC.
Keywords: breast cancer, early diagnosis of breast cancer, microRNA-155
|How to cite this article:|
Metawae E, Abd-Alhamid N, Abd-Allatif S, Abd-Elghaffar N, El-Ghoroury E, Abd-Elrahman A, Ashmawy I, Aboafya Z. Evaluation of microRNA-155 as a diagnostic serum-based biomarker in patients with breast cancer. Al-Azhar Assiut Med J 2018;16:81-6
|How to cite this URL:|
Metawae E, Abd-Alhamid N, Abd-Allatif S, Abd-Elghaffar N, El-Ghoroury E, Abd-Elrahman A, Ashmawy I, Aboafya Z. Evaluation of microRNA-155 as a diagnostic serum-based biomarker in patients with breast cancer. Al-Azhar Assiut Med J [serial online] 2018 [cited 2020 Jul 11];16:81-6. Available from: http://www.azmj.eg.net/text.asp?2018/16/1/81/244147
| Introduction|| |
The most common cancer among Egyptian women is breast cancer (BC), accounting for 32.0% of all cancers in women according to the data of the National Cancer Registry Program of Egypt . However, most of these patients are diagnosed at advanced disease stages: 45.93% at stage III and 15.95% at stage IV . The survival rates improve dramatically when BC is diagnosed early, reaching 90% 5-year survival rate versus 15% for advanced stage of disease . Although most studies focus on improving prognosis with better therapies, a more promising approach is to detect cancer at an early stage. Although the current standard diagnostic imaging tools, mammography and ultrasound, have been successful in detection of early stages of the disease , still there is no sensitive screening serum marker for early detection of BC .
MicroRNAs (miRNAs) are small noncoding RNAs involved in cell differentiation, development, and apoptosis through altering the expression of their target genes . MirRNAs are overexpressed in a number of neoplastic diseases including BC . The expression level of miRNA-155 is up regulated in BC, with high levels associated with clinicopathologic status, tumor subtype, and poor survival rate . Expression levels have been positively associated with metastasis and invasive properties of BC, and increased miRNA-155 serum levels was associated with advanced stage, high tumor grade, and lymph node (LN) metastasis . MiRNA-155 known to play a significant role in the process of carcinogenesis, acting predominantly as an oncomir .
MiRNA-155 suppresses apoptosis in BC cells. Its role in apoptosis could be responsible for its oncogenicity through the blockade of caspase-3 activity . Overexpression of miRNA-155 is followed by a substantial decrease of tumor protein 53-induced nuclear protein 1 which has the ability to induce cell cycle arrest and apoptosis through caspase-3 activation . In our study, we try to evaluate the role of miRNA-155 in early diagnosis of BC in Egyptian female patients.
| Patients and methods|| |
The study was conducted on 50 women divided into two groups: group A included 30 patients with BC admitted to Bab El-Shareya University Hospital from January 2016 to March 2017 (patient group), whereas group B included 20 age-matched and sex-matched normal healthy adult women (control group). The study has been approved by the ethical committee of the National Research Centre, which is in accordance with the ethical standards of the Declaration of Helsinki. In addition, a written informed consent was obtained from all participants before enrollment in the study.
Patients selected in this study had early invasive BC (T1-2, N0-1, M0) , whereas patients with metastatic BC, in-situ tumor, prior history of exposure to radiation, chemotherapy, or hormonal therapy, chronic inflammatory disorders, or other malignancies were excluded. All patients were subjected to the following: full history taking, complete breast examination, breast ultrasound and mammography, chest radiography, abdominopelvic ultrasound, and bone scan. Pathologic diagnosis of the patient’s group using true-cut needle biopsy was done for assessment of pathologic type, grade, and hormone receptors [estrogen receptor (ER) and progesterone receptor (PR)]. Surgical management of the patients was in the form of modified radical mastectomy or conservative breast surgery followed by postoperative pathologic staging.
Overall, 3 ml of venous blood was taken from each women participating in the study under complete aseptic condition and delivered into plain vacutainer with gel separator. The blood was left to clot, and serum was separated by centrifugation at 3000g for 10 min. Immediate extraction followed by reverse transcription of miRNA was performed.
MiRNA was isolated from sera by using the miRNeasy mini kit (cat. no. 217004; QIAgen, GmbH, Hilden, Germany) according to manufacturer’s instructions. Then finally, miRNA was recovered in 30 μl of RNase-free water.
Reverse transcription and quantitative real-time polymerase chain reaction 
Reverse transcription (RT) and quantitative real-time PCR kits (made specifically for accurate miRNA analysis) were used to evaluate the expression of miRNA-155 from serum samples. RT reactions were performed using the TaqMan MicroRNA Reverse Transcription Kit (cat. no. 4366596; Applied Biosystems, Foster City, CA, USA) in a final volume of 15 μl (incubated for 30 min at 16°C, 30 min at 42°C, and 5 min at 85° C and then maintained at 4°C). For real-time PCR, 2 μl of RT products was mixed with 10 μl of Taqman PCR master mixture (No UNG) (cat. no. 4440043; Applied Biosystems, Foster City, CA, USA), 1 μl of TaqMan MicroRNA Assay (cat. no. 4427975; Applied Biosystems, Foster City, CA, USA), and 7 μl of nuclease-free water in a final volume of 20 μl. All reactions were performed on a Quantstudio 12k flex real-time system (Applied Biosystems) with the following conditions: 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. Relative expression of target miRNAs was normalized to miRNA-RNU6B. The fold changes of candidate miRNAs expression were calculated by the equation 2-ΔΔCt.
All results were analyzed using statistical package for the social sciences, IBM SPSS version 20 (Chicago, IL, USA). For nonparametric data, number, percent, quantitative data, mean, SD, and interquartile ranges were used. For comparison of qualitative data, χ2 test, Mann–Whitney test, analysis of variance F test, and Student’s t test were used. P values more than 0.05 were considered to be statistically nonsignificant. P value less than 0.05 was considered significant and P value less than 0.001 was considered highly significant.
| Results|| |
This study was conducted on 30 adult females newly diagnosed with BC, and 20 age-matched healthy women served as a control group.
In this study, 76.7% of the patients were more than or equal to 45 years, 40% were hypertensive, 30% were diabetic, 56.7% were menopausal, and patients with a history of breast feeding represented 86.7%. Regarding the preoperative histopathological results, most of the patients (90%) presented with invasive ductal carcinoma, whereas the rest (10%) presented with invasive lobular carcinoma. Overall, 70% of the patients had grade II BC, whereas 30% had grade III ([Table 1]). According to the postoperative histopathological results of axillary dissection, BC cases were divided into positive axillary LN metastasis in nine cases, and 21 cases showed no LN metastasis. Moreover, 73.3% were ER positive, 26.7% were ER negative, 66.7% were PR positive, and 33.3% PR negative ([Table 1]).
[Table 2] shows a nonsignificant statistical difference between miRNA-155 expression and different parameters of studied patients with BC such as age, menopause, diabetes mellitus, hypertension, breast feeding, histopathology type, grade, and hormonal receptors, whereas positive axillary LNs showed a significant statistical difference (P=0.00013).
|Table 2 Comparison between microRNA-155 expression levels regarding different parameters of studied patients with breast cancer|
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Regarding the assessment of miRNA-155 in all groups, miRNA-155 expression was up-regulated in serum of patients with BC compared with control group. Serum miR-155 expression significantly differed with cancer stage, and expression directly increased with advancing cancer stage.
A comparative study of the miRNA-155 expression between control group and positive and negative metastatic LN subgroups demonstrates a highly significant difference, being the highest in positive LN metastatic subgroup (19.6 fold), followed by negative LN metastasis subgroup (4.2 fold), and lowest in control group (1.06 fold) (P<0.001) ([Table 3] and [Figure 1]).
|Table 3 Comparison between microRNA-155 expressions in all studied groups (mean+SD)|
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|Figure 1 MiRNA-155 expression data among studied groups. 0, negative LN metastasis; 1, positive LN metastasis; LN, lymph node; miRNA, microRNA; N, control group.|
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| Discussion|| |
It is crucial to make a revolution in early detection of BC, which depends on subjective factors like higher education among patients leading breast self- or available regular mammography investigations. Additionally, it is difficult to diagnose BC in its early stage through histological evaluation, so serological biomarkers may serve as an ideal cancer detection tool. Numerous studies have supported the promising role of miRNAs in cancer detection . The role of miRNA-155 in BC highlights a new research focal point for diagnostic and therapeutic plans .
In this study, we investigated the role of miRNA-155 as a diagnostic noninvasive biomarker for early detection of BC. A case–control study was carried out among 30 women diagnosed as having BC by radiology and histopathology, who were compared with 20 healthy women as a control group. Our work revealed that the expression of miRNA-155 significantly differs between groups; it was higher in cases of BC without LN metastasis (stage I) than in control group (4.2 fold). Additionally, the expression was the highest in cases of BC with LN metastasis (stage II) in comparison with both control and stage I groups. In consistence with these results, Lu et al.  noted that high levels of plasma miRNA-155 were identified in the tumors of TNM stage II, LN metastasis 0–3, and tumor sizes of 2–5 cm . Moreover, our results agreed with Sochor et al.  who noted that miRNA-155 elevated in patients with early stage BC compared with healthy participants. Additionally, they concluded that following surgical resection, the serum levels of miRNA-155 significantly decreased . In consistent with these findings, Mar-Agulair et al.  found that the levels of miRNA-155 were significantly higher in patients with BC than in controls .
To understand the role of miRNA-155 in BC requires the identification of critical miRNA-155 targets. A number of conserved targets of miRNA-155 in human genes were identified. Some of these target genes have a confirmed role in BC development such as those encoding SOCS1 and FOXO3a. SOCS1 expression is inversely correlated with miRNA-155 in BC cell lines as well as in a subset of primary breast tumors. Overexpression of miRNA-155 in BC cells induces the activation of transcription 3 (STAT3) and stimulation of BC cells by the inflammatory cytokines interferon-γ, interleukin-6, and lipopolysaccharide, suggesting that miRNA-155 may serve as a bridge between inflammation and cancer .
Accumulated evidence demonstrated that miRNA-155 is usually upregulated in metastatic BC cases ,. Moreover, Eichelser et al.  have identified miRNA-155 as a potential differentiator between metastatic and nonmetastatic BCs . These results agreed with our results as we found that there is a significant difference between cases of BC without LN metastasis and the metastatic ones. A number of studies referred that overexpressed circulating miRNA-155 had its role in breast carcinogenesis ,,.
We analyzed the relation between expression of miRNA-155 and the clinicpathological criteria of the studied patients, in which no significant relation was found except for the positivity of axillary LNs. Our result agrees with Hagras et al.  and Liu et al. , who noted a nonsignificant effect of age, hormonal status, menopausal status, or grading of the tumor on the level of miRNA-155 expression ,. Similarly, Tan et al.  noted that age was not a predictor of miRNA-155 expression, whereas lower differentiation grade, metastasis, and advancement of TNM stage were the only factors that affected expression significantly . However, other patient characteristics that were found by Guo et al.  to be associated with overexpression of miRNA-155 were age of menarche (>13 years), increased times of abortion, lower BMI, and advanced stage of the tumor . Moreover, Nassar et al.  found that the expression of miRNA-155 in Lebanese women was significantly upregulated in age group greater than 40 years, postmenopausal period, and PR positive samples . On the same track, Hafez et al.  and Hamam et al.  noted higher expression levels of miRNA-155 in both ER-positive and PR-positive groups. Chen et al. , Hafez et al. , and Hamam et al.  clarified increased miRNA-155 expression in high tumor grade, advanced stage, and LN metastasis .In the current study, we found that serum miRNA-155 expression was a promising diagnostic and even a prognostic marker in early diagnosis of BC. A mean concentration of 1.94 U/ml was set as the critical point for relative serum miRNA-155 expression of patients with BC. This is in concordance with the study done by Guo et al. , which found that diagnosis of patients with BC is highest when serum concentration is 1.24 U/ml . However, Hamam et al.  and Su-Ying et al.  concluded that expression of miRNA-155 was significantly higher in serum of early-stage BC in comparison with healthy control (reaching to 2.5 fold).
Indeed miRNA-155 is not only a good biomarker for early detection but also the declining levels of serum miRNA-155 after surgery and chemotherapy raise the possibility of its value as an indicator for treatment response . Although many studies revealed the role of miRNA-155 as a biomarker of early BC detection, there are some limitations that may hinder its use, for example, the lack of assurance for accurate measurement and different normalization strategies. In addition, different levels of miRNA-155 have been observed in relation to race, specimen types, and treatment status . Other limitation such as low abundance of miRNA-155 may impede its detection when using standard profiling techniques such as microarray .
| Conclusion|| |
The expression of miRNA-155 was higher in patients with BC compared with controls, with higher expression in those with metastasis. So these results validate miRNA-155 as a promising diagnostic and even a prognostic marker in cases of BC. Detection of serum miRNA-155 may become an important noninvasive screening test for BC in the future. However, our sample size was small, so we recommend increasing the number of participants to enhance validity of the results.
This study was funded by National Research Centre, Egypt.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest
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[Table 1], [Table 2], [Table 3]