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Latin American Journal of Biotechnology and Life Sciences
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INVESTIGATION / RESEARCH

Upregulation of miR-206 is a potential diagnostic biomarker in breast cancer

Faezeh Karami 1*#, Narges Maleki 1,2, Arefeh Khazraei Monfared 1, Sayeh Jafari Marandi 1
Available from: http://dx.doi.org/10.21931/RB/2021.06.02.15

 
ABSTRACT

Breast cancer is one of the most common malignancies, and like most cancers, most cases are caused by somatic mutations. Due to estrogen's role in the growth, differentiation, and division of breast and endometrial cancer cells, tamoxifen is used as an estrogen receptor antagonist in breast cancer cells with estrogen receptor (ER +) has a special place, which unfortunately in one-third of the Cases are resisted. This study aimed to investigate the effect of tamoxifen-treated tumor-derived exosomes on the expression pattern of Twist and Bcl-2 oncogenic genes in fibroblast cells. MCF-7 breast cancer cell line and fibroblast cells were purchased and cultured in a complete culture medium. After the appropriate number of cells was reached, they were treated with the appropriate concentration of tamoxifen. Cellular supernatant was then gathered in flasks, and exosomes were extracted from them. After extracting RNA from exosomes and cDNA synthesis, the expression level of miR-206, Twist-1, and Bcl-2 genes were evaluated using the Real-Time PCR method. The electronic microscope results confirmed the correctness of the exosomes isolated from the tumor cell culture medium. It has also been shown that tamoxifen treatment increases the expression of miR-206 in exosomes derived from breast tumor cells. The control group which has been kept untreated induced the expression level of Twist-1 and Bcl-2 genes time-dependently. However, when tamoxifen-treated tumor-derived exosomes treated the target cells, the expression level of oncogenic miRs Twist-1 and Bcl-2 were declined over time. Overall, this study showed that tamoxifen treatment on breast cancer cells could apply its antioncogenic effects on tumor stromal cells, such as fibroblasts, by altering the expression levels of exosomal microRNAs in tumor cells.

Keywords: breast cancer, tamoxifen, fibroblast, exosome, miR-206

 
 
INTRODUCTION
 
Breast cancer is one of the most common malignancies, and like most cancers, most cases are caused by somatic mutations. Today, breast cancer is the most common cancer among women globally, and the resulting death rate is the highest in cancer deaths among women 1. Tamoxifen is a non-steroidal anti-estrogenic drug introduced in the United Kingdom in 1960 as a contraceptive drug and was approved by the Food and Drug Administration in 1977 as a treatment for metastatic breast cancer. The adjuvant therapy title for breast cancer is used in people at risk 2. As a treatment for breast cancer, tamoxifen slows or stops the growth of cancer cells that need estrogen to grow or spread, and as a complementary treatment, it helps prevent the spread and recurrence of breast cancer 3. The exact mechanism of action of the drug is unclear. Tamoxifen may act by blocking estrogen receptors in tumor cells that need estrogen to grow. The estrogen receptor- tamoxifen complex may be transported into the nucleus of a tumor cell, inhibiting DNA production 4.
Exosomes are vesicles at the nanometers that are actively secreted by almost all types of cells, including fibroblasts, endothelial cells, epithelial cells, nerve cells, immune cells, and cancer transmit signals between cells. This suggests that exosomes play several roles in regulating physiological responses 5. The pathophysiological effects of exosomes on diseases, especially cancers, have recently been identified. According to research, tumor-derived exosomes play a role in the spread of cancer malignancy by increasing cancer cells' proliferation, creating a pre-metastatic site, and regulating drug resistance. Clinically, exosomes' performance as diagnostic biomarkers, therapeutic targets, or even as carriers of anticancer drugs has been emphasized due to their unique biological and pathophysiological characteristics 6.
MiRNAs are small molecules that play a crucial role in physiological, pathological, angiogenesis, apoptosis, and cancerous processes. Today, miRNA is used as a biomarker to diagnose and treat cancer to prevent cancer patients' difficult stages of treatment. A miRNA is a short ribonucleic acid (RNA) found in eukaryotic cells. A miRNA molecule has a small number of nucleotides than other RNAs (it has an average of 22 nucleotides). MiRNAs are post-transcription regulatory elements that bind to a complementary sequence on the target mRNA, resulting in suppressing the transcription, degrading the target, and ultimately silencing the gene. The human genome encodes more than 2,000 types of miRNAs that may make up 60% of genes and are abundant in various human cells 7. The TWIST1 gene encodes a transcription factor 8, and the BCL2 gene encodes an anti-apoptotic protein 9. Based on what has been previously saying and the hypothesis that tumor cell secretions could cause malignancy in the fibroblast cells of the tumor microenvironment, so in the present study, we sought to investigate the effect of tumor cell-derived exosomes on the expression level of Twist-1 and Bcl-2 oncogenes as well as miR-206 in tumor cells.

 
MATERIALS AND METHODS
 
Cell Purchase and preparation
 
MCF-7 breast cancer cell line with NCBI CODE 135 was purchased from Iran Genetic Resources Center, and fibroblast cells were purchased from Tarbiat Modares University. The cells were stored in a fully cultured medium with FBS, and were kept in an incubator with CO2 Saturation Rate: 5% and a temperature of 37C.
 
 
Tamoxifen treatment
 
Based on the results of the previous experiments 10, tamoxifen IC50 was a concentration between 54μM (equivalent to 20μg/ml) and 68μM (equivalent to 25μg/ml). Hence, the concentration of 60Mμ (as a concentration close to IC50) was considered for this study's treatment.
 
 
Exosome Isolation
 
To isolate the exosomes from the culture medium of MCF-7 cells, centrifugation at 3000g was performed for 10minutes, and an exoquick solution (system biosciences) was added to the liquid. The microtubes were incubated at 4C for 20min and then centrifuged again at 1500g for 15min. 50 microliters of PBS were added to the resulting precipitate. Eventually, the microtubes were stored at -20C. The exosome isolation from the supernatant was performed on both treated and control cell groups. The exosomes' extraction was confirmed by microscopic examination, and the size and morphology of the exosomes were evaluated by a digital electron microscope (Digital FESEM, KYKY-EM3200, China).
 
 
Labeling Exosomes
 
The isolated exosomes were labeled using PKH26 (PKH26 Red fluorescent - Mini26-1, Sigma) dye. Initially, one milliliter of Diluent C was added to one milliliter of the exosomes. After pipetting, one milliliter of diluted dye was added to the microtube. After incubation at room temperature for 5 min, two milliliters of Diluent C were added to the microtubules to stop labeling. In the next step, 1.5ml of exoquick, exosome separation solution, was added with a ratio of 3 to 1 (relative to the content of microtubes), and after incubation for 20 minutes at 4C, centrifugation at 15000g was done for 20 minutes. The sediment was then dissolved in 30 microliters of PBS. Fibroblast cells were treated with these labeled exosomes and incubated at room temperature for 12 hours. Due to miRNAs' presence in exosomes, the exosomal transfer of miR-206 to fibroblast cells was investigated. In this regard, fibroblast cells were treated with 100µg/ml tumor-derived exosomes and 50µg/ml alpha-amanitin to evaluate the miR-206 transfer rate fibroblast cells at 0, 24, and 48 hours after treatment. The reason for using alpha-amanitine is to inhibit any transcription activity that can be induced by exosomal treatment in cells.
 
 
RNA Isolation
 
Initially, 50 microliters of trizol (Invitrogen) were added to the cells. They were then incubated at room temperature for 5 min, and then 15μl of chloroform were added to them, and the microtubes were incubated at room temperature for 2-3 min. The sample microtubes were centrifuged at 12000g for 10minutes. At this stage, the upper solution was transferred to a new microtube as RNA. One milliliter of ethanol 75% was added and then pipetted. Finally, the microtubes were stored at -80C. The extracted RNA's quantitative assay was performed using spectrophotometry (NanoDrop Technologies, Wilmington, DE, USA).
 
Evaluating the expression level of TWIST1, BCL2, and miR-206 genes: For cDNA synthesis, TWIST1 and BCL2 genes were used (1st strand cDNA Synthesis kit - Thermo Fisher Scientific). Moreover, oligo dT, primer, and random hexamer were all added with one ratio, and the synthesis was continued according to the manufacturer's guidelines. Thermofisher kit and the Aligo dT (Exiqon) kit were used to evaluate the expression of miR-206. The sequence of primers used for each gene in the Real-time PCR reaction is listed in Table 1. The temperature cycle was similar for all three genes and began with an initial eruption at 95 C for 5 min, then with 40 cycles, each involving refraction at 95 C for 5 s. The connection process was maintained at 65-62. C for 20 s and the expansion at 72. C for 30 s. Data analysis was performed using the CT comparison method. Compared to the endogenous U6 snRNA expression levels and expression of other genes, normalization of miRNA expression levels was normalized and compared by GAPDH gene. Data analysis was performed using ABI step one Real-Time PCR Software v2.0.2 software (Applied Biosystems, UK) and statistical analysis using Excel software (Microsoft, 2010) and GraphPad software.
 


 
Table 1. The sequence of primers used in the real-time PCR reaction
 
 
Statistical analysis: The data obtained from the present study were analyzed using SPSS v.20 software and one-way and Tukey variance statistical tests, and P <0.05 was considered significantly meaningful.

 
 
RESULTS

 
Exosome confirmation
 
To investigate the exosomes' molecular content derived from breast tumor cells, the exosomes were first isolated from the tumor cells' culture medium by the method mentioned above. The first is to confirm these exosomes by any appropriate methods that assess the exosome's size and morphology. The size and morphology of the exosomes were evaluated by electron microscopy. The results showed that the isolated exosomes had a spherical appearance with a size range of 30 to 150 nm (Figure 1).
 


 
 
Figure 1. Examination of exosomes isolated by electron microscopy. The exosomes' examination using electron microscopy showed the spherical appearance and size of 30-150 nm of the exosomes.

 
 
Cellular uptake of PKH26-labeled fluorescent colored tumor exosomes to fibroblast cells
 
To determine tumor cell-derived exosomes' ability to enter fibroblast cells, fPKH26-labeled fluorescent colored tumor exosomes were incubated with fibroblast cells for 12 hours. Fluorescent microscopic analysis showed that PKH26-labeled fluorescence-labeled exosomes are located in the cytoplasm of fibroblast cells (Figure 2). This indicates the ability of fibroblast cells to absorb exosomes derived from tumor cells.
 
 
 

 
Figure 2: Left: Morphological view of fibroblastic cells with spindle-shaped appearance (magnification x90). Right: PKH26-labeled tumor exosomes located in the cytoplasm of fibroblast cells

 
 
Effects of exosomal treatment on fibroblast cells and the transfer of exosomal miR-206 to fibroblast cells
 
As seen in Figure 3, levels of miR-206 transcripts in fibroblast cells treated by exosomes and alpha-amanitin showed a significant increase over time.


 
 
Figure 3. The expression level of miR-206 being treated by tamoxifen. Examining the miR-206 expression level at 0, 24, and 48 hours showed that the gene expression level increased significantly in a time-dependent manner.  

 
 
Examination of miR-206 expression levels in breast cancer cell-derived exosomes in the presence and absence of tamoxifen
 
MiR-206 expression levels in tumor cell-derived exosomes treated by tamoxifen have higher expressions than those of the control group (Figure 4). This result suggests that tamoxifen treatment may alter the expression level of miR-206 in tumor cell-derived exosomes.


 
 
Figure 4. Expression levels of miR-206 in tumor cell-derived exosomes treated by tamoxifen compared with the control group. This gene expression in exosomes derived from tamoxifen-treated cells is significantly higher than those of the control group.

 
 
Effect of breast tumor-derived exosomes on the expression level of Twist-1 and Bcl-2 genes in fibroblastic cells
 
Tamoxifen-Treated tumor-derived exosomes induce the Twist-1 gene expression time-dependently; However, tumor-derived exosomes that had been left untreated reduced the gene expression of Twist-1 over time (Figure 5).


 
 
Figure 5. The inhibitory effect of tamoxifen-treated tumor-derived exosomes versus the progressive effect of untreated treated tumor-derived exosomes on the expression of the Twist-1 gene over time. The GAPDH is used for normalization. The expression of the TWIST-1 gene being treated with the drug exosome (+) has a down-regulation and has increased over time, while the expression of the TWIST-1 gene in drug (-) treated exosome cells has increased over time.

 
 
Regarding the Bcl-2 oncogene, the results showed that when tumor cells were treated with tamoxifen, exosomes derived from these types of cells reduced Bcl-2 expression over time. However, exosomes derived from tumor cells that did not receive medication had an increasing effect on the expression of the Bcl-2 gene (Figure 6).


 
 
Figure 6. The inhibitory effect of tamoxifen-treated tumor-derived exosomes versus the progressive effect of untreated treated tumor-derived exosomes on the expression of the BCL-2 gene over time. The GAPDH is used for normalization. The expression of the BCL-2 gene being treated with the drug exosome (+) has a down-regulation and has increased over time, while the expression of the BCL-2 gene in drug (-) treated exosome cells has increased over time.

 
 
DISCUSSION

 
Past studies have shown that exosomes are rich in microRNAs 11, so it seems that the exosomal transfer of miRNAs from tumor cells could be a factor in cell malignancy. It can also be inferred that pharmacological agents can alter tumor exosomes' molecular contents and prevent tumor formation. The present study showed that tamoxifen treatment increased miR-206 expression in tumor cell-derived exosomes. Previous studies have shown that miR-206 acts as a suppressor for tumors by directly targeting transcripts of the Twist-1 gene in breast cancer's pathogenesis and that its expression is inversely related to tumor development 12. Afterward, this paper examined the exosomal transfer of miRNA-206 to fibroblast cells that had been treated with tumor exosomes (treated with tamoxifen). This study hypothesized that tamoxifen treatment on the tumor cell could alter the selected miRNA expression pattern in tumor cell exosomes. So, in this study, the expression level of miR-206 in cell-derived exosomes was also assessed. MCF-7 breast tumor cell line was categorized into two groups: the experiment group that received tamoxifen and the control group that did not receive medication. Subsequently, the experiment group was treated with tamoxifen. Expression levels of miR-206 in tumor cell-derived exosomes treated with tamoxifen showed higher expression than in control cell-derived exosomes. This result suggests that tamoxifen treatment may alter the expression of miR-206 in tumor cell-derived exosomes.
 
In the tumor microenvironment, tumor cell secretions can cause malignancy to their microbial fibroblasts; so, in the present study, we scrutinized the effect of tumor cell-derived exosomes on the expression of Twist-1 and Bcl-2 oncogenes. Much to our surprise, untreated tumor-derived exosomes induced the expression of the Twist-1 gene time-dependently, while the experiment group reduced the expression of Twist-1 over time. Therefore, given the more significant presence of miR-206 in tamoxifen-treated tumor-derived exosomes, it is concluded that the transferring of the miR-206 was the underlying reason for the decrease in the expression level of Twist-1 over 48 hours of assessment. The Bcl-2 oncogene followed precisely the same expression pattern as Twist-1. In other words, untreated tumor-derived exosomes induced the expression of the BCL-2 gene time-dependently, while the experiment group reduced the expression of BCL-2 over time. Overall, the present study suggests that miR-206 exosomal transferring from mammary tumor cells treated with tamoxifen may have inhibitory effects on oncogenesis through diminishing the activity and expression of Twist-1 and Bcl-2 genes in fibroblast cells.
 
They are comparing gene expression profiles 13. It is reported that increased Twist-1 gene expression was associated with invasion and metastasis of breast cancer. Suppression of Twist-1 expression by siRNA in metastatic breast carcinoma cells specifically inhibits cells' ability to metastasize from the mammary gland to the lung. The expression of Twist-1 in epithelial cell lines leads to loss of cell-to-cell junction through E-cadherin, activation of mesenchymal markers, and induction of cell movement; Twist-1 binds to E-box elements in the promoter region of E-cadherin, the transcription expression of which suppresses cell-to-cell junction. Based on these findings and knowing the function of Twist-1 as a significant regulator in fetal formation, the researchers hypothesized that Twist-1 would help metastasis by increasing the EMT stage in cancer progression 14. Therefore, a decrease in the expression of Twist-1 under the influence of miRNA-206 can indicate the function of the miRNA-206 as a tumor suppressor.
 
On the other hand, the high expression of miR-206 in exosomes derived from tamoxifen-treated cells suggests how this drug's use may affect the expression of Twist-1 and Bcl-2. In fact, with the use of tamoxifen, the expression of miR-206 in the exosomes increases, which in turn reduces the expression of Twist-1 and Bcl-2, resulting in a decrease in cancer cell metastasis. In 2016, Zhong et al. examined the expression profile in drug-resistant breast cancer cells and their exosomes using microsurgery. Their results showed that most miRNAs in exosomes had low expression over cells. However, some miRNAs were more expressed in exosomes, indicating that some miRNAs were concentrated in exosomes. They found 22 miRNAs overexpressed in exosomes and cancer cells themselves, and 12 miRNAs increased that expression after chemotherapy 13.
 
The present experiment results generally showed that when breast cancer cells were treated with tamoxifen, the miR-206 content in these cells' exosomes was higher than that of cancer cells without drug treatment. Since exosomes are a rich and protective source for miRNAs and transfer miRNAs between cells in the tumor microenvironment, treatment of tumor exosomes on fibroblast cells causes miRs' transferring. Untreated tumor-derived exosomes increased the expression of Twist-1 and Bcl-2 genes in a time-dependent manner in fibroblast cells. This could be the reason for the malignancy of fibroblastic cells in the vicinity of tumor cells. In contrast, tamoxifen-treated tumor-derived exosomes reduced the expression of Twist-1 and Bcl-2 genes over time in fibroblast cells. This may be because the therapeutic effect of tamoxifen in breast cancer patients is due to its effect on exosomal miRNAs' expression, such as miR-206, and its consequent reduction in tumor progression.

 
 
CONCLUSIONS
The present experiment results generally showed that when breast cancer cells were treated with tamoxifen, the miR-206 content in these cells' exosomes was higher than that of cancer cells without drug treatment. Since exosomes are a rich and protective source for miRNAs and transfer miRNAs between cells in the tumor microenvironment, treatment of tumor exosomes on fibroblast cells causes miRs' transferring. Untreated tumor-derived exosomes increased the expression of Twist-1 and Bcl-2 genes in a time-dependent manner in fibroblast cells. This could be the reason for the malignancy of fibroblastic cells in the vicinity of tumor cells. In contrast, tamoxifen-treated tumor-derived exosomes reduced the expression of Twist-1 and Bcl-2 genes over time in fibroblast cells. This may be because the therapeutic effect of tamoxifen in breast cancer patients is due to its effect on exosomal miRNAs' expression, such as miR-206, and its consequent reduction in tumor progression.


 
 
Conflict of interests
 
No. The authors declare that they have no competing interests.
 
Availability of data and materials
 
The data used in this study are available from the corresponding author on request.
 
Consent for publication
 
All the authors confirm that the manuscript represents their honest work.

 
 
 
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Received: 15 December 2020
 
Accepted: 14 February 2021
 
 
 
Faezeh Karami 1*#, Narges Maleki 1,2, Arefeh Khazraei Monfared 1, Sayeh Jafari Marandi 1
1. Department of Cellular and Molecular Biology, Faculty of Biological Sciences, Islamic Azad University-Tehran North Branch, Tehran, Iran. P.O. Box: 19585/466, Telephone: +98212565149, Fax: +982177009847;
2. Gynecology and Reproductive Biology Department, Kowsar poly-clinic, Tehran, Iran. P.O box: 1658143443, Telephone:  +982176492539, Fax:  +982176492539 extension 6
* Corresponding authors:
* Faezeh Karami. Department of Cellular and Molecular Biology, Faculty of Biological Sciences, Islamic Azad University-Tehran North Branch, Tehran, Iran. P.O. Box: 19585/466 , Telephone: +98212565149, Fax: +982177009847. Email: karami_faezeh@yahoo.com - https://orcid.org/0000-0002-7779-748X
ORCID
Faezeh Karami  https://orcid.org/0000-0002-7779-748X
Narges Maleki  https://orcid.org/0000-0002-2702-0015
Arefeh Khazraei Monfared https://orcid.org/0000-0002-7503-3179
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