|Year : 2021 | Volume
| Issue : 2 | Page : 91-96
Circulating serum micro-ribonucleic acids 155 and 205 before and after surgery in patients with breast cancer
K Rama1, Aparna R Bitla1, Narendra Hulikal2, Mutheeswaraiah Yootla3, Lakshmi Amancharla Yadagiri4, T Asha5, M Manickavasagam6, PV L. N. Srinivasa Rao1
1 Department of Biochemistry, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India
2 Department of Surgical Oncology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India
3 Department of Surgery, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India
4 Department of Radiology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India
5 Department of Pathology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India
6 Department of Medical Oncology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India
|Date of Submission||08-May-2020|
|Date of Acceptance||02-Feb-2021|
|Date of Web Publication||17-Jul-2021|
Aparna R Bitla
Professor, Department of Biochemistry, Sri Venkateswara Institute of Medical Sciences, Tirupati - 517 507, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
Background: Micro-ribonucleic acids (miRNAs) are small non-coding RNAs that regulate gene expression. Expression of miRNAs has been shown to be dysregulated in patients with breast cancer. These markers have been shown to be potential markers of diagnostic significance. We intended to study the usefulness of miRNA-155 and miRNA-205 in monitoring response to surgery by monitoring their pre- and post-operative expression.
Methods: Twenty patients newly diagnosed with malignant breast disease along with 20 age-matched females not having any breast lesion were included in the study. Reverse transcription–polymerase chain reaction was used to assess the expression of circulating miRNAs.
Results: MiRNA-155 was found to be significantly upregulated and miRNA-205 was significantly downregulated in patients with breast cancer compared to controls. There was no change in expression of miRNA-155 and miRNA-205 following surgery.
Conclusions: The findings of the present study do not support the role of miRNA-155 and miRNA-205 as markers suitable for follow-up. However, the findings of the present study need to be confirmed in a larger sample size and samples collected over a defined post-operative period.
Keywords: Breast cancer, follow-up, micro-ribonucleic acid-155, micro-ribonucleic acid-205, surgery
|How to cite this article:|
Rama K, Bitla AR, Hulikal N, Yootla M, Yadagiri LA, Asha T, Manickavasagam M, L. N. Srinivasa Rao P V. Circulating serum micro-ribonucleic acids 155 and 205 before and after surgery in patients with breast cancer. J Clin Sci Res 2021;10:91-6
|How to cite this URL:|
Rama K, Bitla AR, Hulikal N, Yootla M, Yadagiri LA, Asha T, Manickavasagam M, L. N. Srinivasa Rao P V. Circulating serum micro-ribonucleic acids 155 and 205 before and after surgery in patients with breast cancer. J Clin Sci Res [serial online] 2021 [cited 2021 Aug 3];10:91-6. Available from: https://www.jcsr.co.in/text.asp?2021/10/2/91/321699
| Introduction|| |
With the recent data showing around 14 million new cases and 8.2 million cancer-related deaths worldwide breast carcinoma the most common cancer in women. Biomarkers play an important role in diagnosis, prognosis and monitoring response to treatment of any disease. At present, cancer antigen 15-3 and carcinoembryonic antigen are most commonly utilised biomarkers in monitoring treatment response and prognosis of the metastatic breast cancer. Micro-ribonucleic acids (miRNAs) are small, non-coding RNAs that regulate gene expression in a sequence-specific manner. These have been identified as biomarkers in many diseases including many cancers. A miRNA that is downregulated in cancer and targets an oncogene might normally function as a tumour suppressor, whereas an upregulated miRNA which targets a tumour suppressor might act as an oncogene.
Studies have shown that miRNAs are dysregulated in many malignancies including breast cancer. The aberrantly expressed miRNAs have been shown to be of potential diagnostic, prognostic and therapeutic use. miRNA-155 is a tumour-promoting miRNA and plays an important role in various physiological and pathological processes and is the most frequently upregulated oncogenic miRNA reported along with miRNA-21 in breast tumour tissue., miRNA-205 has been shown to be downregulated in breast cancer. There is one Indian study on the role of miRNA-21 in patients with breast cancer. With this background, the present study was taken up to study the expression of one upregulated miRNA (miRNA-155) and one downregulated miRNA (miRNA-205) in patients with breast cancer and assess the effect of surgery on the expression of these miRNAs.
| Material and Methods|| |
The present study included 20 newly diagnosed patients with malignant breast disease (cases) attending the oncology (medical and surgical) and general surgery outpatient departments of a tertiary care centre in South India after written informed consent. Twenty age-matched healthy females not having any breast lesion on physical examination and/or confirmed by mammography were included as controls. The study was approved by the institutional ethics committee. Male breast cancer patients; patients having tumours other than breast; women on hormone replacement therapy; those who are using oral contraceptive pills; those with active inflammation, diabetes, hypertension and thyroid disorder and those not willing to participate in the study were excluded from the study.
Following overnight fasting, 5 mL of peripheral venous blood was drawn into a serum separator tube (BD Vacutainer, Hyderabad, India, cat no: 367958) from the participants before surgery. A second blood sample was drawn from breast cancer patients after surgery at the time of discharge between 10 and 20 days. The blood was allowed to clot for 30 min and centrifuged at 3000 rpm for 15 min. The serum was separated, aliquoted and stored at −80°C until RNA extraction. Serum samples were used for studying the expression of miRNA.
miRNA expression was studied using reverse transcription–polymerase chain reaction (RT-PCR). First total RNA was isolated from 600 μL of serum previously thawed on ice using Purefast Total RNA (miRNA) Minispin prep kit (Helini Biomolecules, Chennai, India) according to the manufacturer's protocol. The RNA was eluted with 50 μL of RNAse-free water. The concentration of all RNA samples was quantified using NanoDrop 1000 (Thermo Scientific, Wilmington, DE, USA). miRNA expression levels were quantified using the Roche LightCycler 96 (company Roche, Indianapolis, USA) using TaqMan probe-based miRNA real-time PCR kit (Helini Biomolecules, Chennai, India), target miRNA in FAM channel and heterologous internal control in HEX Channel. Primers used (Helini Biomolecules, Chennai, India) are shown in [Table 1].
Briefly, 20 ng of total RNA was used from each sample for the individual assays in 20 μL reactions containing RT mixture and primers. The mix was incubated at 25°C for 15 min (primer annealing), 42°C for 60 min (RT) and 85°C for 5 min (RT inactivation). For this purpose, 2.5 μL of RT reaction was mixed with 10 μL of probe PCR master mix, 2.5 μL of miRNA-specific primer-probe mix, 2.5 μL of internal control primer-probe mix and 7.5 μL PCR grade water were added to get a final volume of 25 μL. RT-PCR was done in duplicate, including no-template controls. Cel-miRNA-39-3p was used as an endogenous control to normalise the data. The relative expression of the mature miRNA was calculated using the comparative cycle threshold (Ct) (2^ΔΔCT) method, where ΔCT = CT (case/control)−CT (endogenous control) whereas ΔΔCT = ΔCT (case)−ΔCT (control). We used Cel-miRNA-39-3p as an endogenous control to normalise the data. The inter- and intra-assay coefficient of variation for miRNA analysis was <5%.
Data distribution was studied using Kolmogorov–
Smirnov test. Continuous variables are expressed as mean ± standard deviation or median interquartile and as frequency (number [%]) for categorical data. Mann–Whitney U-test was used to study the difference in miRNAs between cases and controls. Paired t-test was used to compare the expression of miRNAs before and after surgery. Data analysis was performed using Microsoft Excel spreadsheet (Microsoft Corporation, Redmond, USA), Statistical Package for Social Sciences (SPSS) for Windows version 11.5 (SPSS Inc., Chicago, IL, USA) and MedCalc statistical software (version 13.2.2, Belgium). A P < 0.05 was considered as statistically significant.
| Results|| |
The demographic data of the study participants are shown in [Table 2]. [Table 3] shows the delta Ct values obtained for miRNA-155 and miRNA-205 (median [interquartile range]) amongst cases and controls. [Table 3] shows the demographic and clinical characteristics of the study population. The groups were matching in BMI and age at first live birth. Breast cancer patients were older than controls (P < 0.05). None of the controls had a family history of breast cancer. Oestrogen receptor/progesterone receptor status was positive in 35% (n = 7) and negative in 57% (n = 11) of the patients with breast cancer. The tumour type was ductal carcinoma in 90% (n = 18) and lobular carcinoma in 10% (n = 2) of the cases. Eight cases (40%) each had Stage I and Stage II A disease while 4 (20%) had Stage II B disease. None had Stage III or IV disease.
|Table 3: Comparison of micro-ribonucleic acids between cases and controls|
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A significant increase in expression of miRNA-155 [Table 3] and [Figure 1] was observed between patients with malignant breast disease (P < 0.05) compared to controls as evidenced by a decrease in delta Ct (higher the expression lower is the Ct) [Table 3]. Expression of miRNA-205 was significantly lower (P < 0.05) in patients with malignant breast disease compared to controls as evidenced by an increase in Ct compared to controls (lower the expression, higher is the Ct) [Table 3] and [Figure 1].
|Figure 1: Box plots showing the comparison of expression of miRNA in patients with malignant breast disease and healthy controls. miRNA-155 (P < 0.05) and miRNA-205 (P < 0.05) delta Ct = Change in cycle threshold; miRNA= micro ribonucleic acid|
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As shown in [Table 4] and [Figure 2], no significant difference was observed in expression of miRNA-155 and miRNA-205 following surgery when compared to their expression before surgery.
|Figure 2: Box plots showing the comparison of expression of miRNA in patients with malignant breast disease before and after surgery. miRNA-155 (P < 0.05) and miRNA-205 (P < 0.05) delta Ct = Change in cycle threshold; miRNA= micro ribonucleic aci|
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|Table 4: Comparison of micro-ribonucleic acid expression levels in patients with malignant breast disease (n = 20) before (Ct) and after surgery|
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| Discussion|| |
Circulating tumour cells in blood have been used for assessment of prognosis in patients with metastatic breast cancer. A significant increase in expression of miRNA-155 was observed in patients with malignant breast disease (P < 0.05) compared to controls as evidenced by a decrease in delta Ct [Table 3]. This is in agreement with previous studies,, reporting a significant increase in serum miRNA-155 expression in patients with malignant breast tumour. miRNA-155 downregulates the suppressor of cytokine signalling 1 in breast cancer, in turn leading to persistent activation of signal transducer and activator of transcription 3 signalling and inflammatory cascades, suggesting a link between inflammation and cancer through miRNA-155. miRNA levels have been reported to be significantly higher in Stage I and II compared to Stage III in breast cancer patients. High expression of miRNA-155 along with miRNA-222 was recently reported to be associated with positive lymph node status, especially in patients with triple-negative BC.
The expression of miRNA-205 was significantly lower (P < 0.05) in patients with malignant breast disease compared to controls as evidenced by an increase in Ct compared to controls (lower the expression, higher is the Ct) [Table 3]. This result is in agreement with previous studies, reporting a significant decrease in the expression of miRNA-205 in patients with malignant breast disease.
A significantly decreased expression in serum miRNA-205 was first reported by Zhang et al. miRNA-205 expression was found to be significantly lower in breast cancer patients compared to healthy controls. Furthermore, the difference between Stage I patients with breast cancer and healthy controls was statistically significant. This shows that miRNA-205 can differentiate between patients with early breast cancer and healthy controls. A decrease in expression has also been reported in patients with inflammatory breast cancer compared to non-inflammatory breast cancer. Lower expression of miRNA-205 was found to correlate with worst distant metastasis-free survival and overall survival.
In a recent study, miRNA-155 was found to be significantly decreased following chemotherapy. This shows its usefulness to assess response to therapy. It has also been shown to be a predictor of early breast cancer relapse along with miRNA-24. miRNA-155 was found to be significantly downregulated in tumour tissues of patients with neoadjuvant chemotherapy (NAC) compared to patients without NAC, thus showing the importance of miRNA-155 and miRNA-222 as molecular markers for prognosticating purpose. We evaluated miRNA-155 expression following surgery. However, we did not observe any change in expression of miRNA-155 following surgery. However, miRNA-155 serum levels were found to be significantly reduced after 3 weeks of surgery compared to their pre-operative levels. The mean miR-155 level of patients with breast cancer tended to be increased on day 3 after surgery with the mean value returning to the pre-operative level on day 7 and then falling to below baseline at day 30. A previous study reported that 90% (n = 26) of all patients showed a significant decrease in levels of miRNA-155 after surgery, with a 73.1% median decrease (range between 10th and 90th percentiles, 10.7%–95.5%), whereas three patients actually showed an increase (106%, 158% and 250%) in their study may lead to contradictory finding. In another study, post-operative samples were collected from 10 breast cancer patients before surgery and after surgery (2 weeks and 6 months), 49% of the total samples showed downregulation, whereas 51% showed upregulation after the surgery.
Similarly, no change in expression of miRNA-205 was seen after surgery [Table 4]. There are no studies on the effect of surgery on miRNA-205 expression in patients with breast cancer. Most downregulated miRNA shows a trend towards the downside and upregulated miRNA shows a trend towards the upper side after the surgery. It is not clear whether the circulating miRNA levels change between 2 and 3 weeks post-surgery. However, it seems likely as miRNAs associated with subclinical inflammation due to the surgery, as well as miRNAs associated with the tumours, would gradually decline.
The possible explanation for no change in expression of miRNA-155 and miRNA-205 in malignant breast cancer patients after surgery in the present study could be the time point studied after surgery. The collection of post-operative sample ranged from 10 to 20 days. Collection of fixed duration samples was not possible due to logistic reasons of patient availability. Differences in the time of collection of the post-operative samples may impact miRNA expression levels.
The findings of the present study do not support the role of miRNA-155 and miRNA-205 as markers suitable for follow-up. This could be due to the time point studied which was between 10 and 20 days. Collection of fixed duration samples was not possible due to logistic reasons of patient availability. The findings of the present study need to be confirmed in a larger sample size with fixed duration sample collection.
Financial support and sponsorship
The study was funded by DST-Inspire Fellowship, New Delhi (DST/INSPIRE Fellowship/2013/IF130888), and 'Sri Balaji Arogya Varaprasadini Scheme' of Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh (SBAVP-RG/Ph.D/15).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]