Breast cancer nipple discharge exosomal microRNAs are stable under degradative conditions Wang YW, Zhang W, Chen X, Tian Y, Zhao S, Zhang K, Zhu J, Ma R, Wang J,

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Breast cancer nipple discharge exosomal microRNAs are stable under degradative conditions

Ya-Wen Wang¹, Weiguo Zhang², Xu Chen³, Yaru Tian⁴, Song Zhao¹, Kai Zhang¹, Jiang Zhu¹, Rong Ma¹, Jianli Wang⁵
¹ Department of Breast Surgery, General Surgery, Qilu Hospital of Shandong University, Jinan, Shandong, China
² Department of Breast Surgery, General Surgery, Qilu Hospital of Shandong University, Jinan, Shandong; Department of General Surgery, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning, China
³ Department of Pathology, Qilu Hospital of Shandong University, Jinan, Shandong, China
⁴ Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong, China
⁵ Department of Obstetrics and Gynecology, Qilu Hospital of Shandong University, Jinan, Shandong, China

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Date of Submission	17-Nov-2022
Date of Decision	22-Feb-2023
Date of Acceptance	13-Mar-2023
Date of Web Publication	28-Apr-2023

Abstract

We have previously shown that microRNAs (miRNAs) in nipple discharge are potential diagnostic biomarkers. In particular, exosomes are present in nipple discharge. Herein, we sought to elucidate the protective role of exosomes on miRNAs in nipple discharge and investigate the stability of miRNAs encapsulated in exosomes under degradative conditions. A novel TTMAAlPc-RNA complex method was used to measure the RNase concentration in colostrum and nipple discharge. Quantitative real-time polymerase chain reaction was performed to test the stability of exogenous synthetic miRNAs (cel-lin-4-5p and cel-miR-2-3p) and endogenous miRNAs (hsa-miR-4732-5p, hsa-miR-3646, hsa-miR-4484, and kshv-miR-K12-5-5p). RNase was present and functional in colostrum and nipple discharge. Endogenous miRNAs were more stably expressed compared to exogenous miRNAs at room temperature and 4°C. Triton X-100 (1%, 30 min) destroyed the exosomal membrane, causing RNA degradation in colostrum but not in nipple discharge. Therefore, we confirmed that exosomes in colostrum and nipple discharge could protect miRNAs from degradation by RNase. Exosomes in nipple discharge may be more resistant to Triton X-100 lysis compared to those in the colostrum. Exosomal miRNAs in nipple discharge in breast cancer are stable under degradative conditions. Differential Triton X-100 sensitivity of exosomes of nipple discharge and colostrum warrants further investigation.

Keywords: Breast cancer, exosome, microRNA, nipple discharge, stability, Triton X-100

How to cite this URL:
Wang YW, Zhang W, Chen X, Tian Y, Zhao S, Zhang K, Zhu J, Ma R, Wang J. Breast cancer nipple discharge exosomal microRNAs are stable under degradative conditions. Chin J Physiol [Epub ahead of print] [cited 2023 May 29]. Available from: https://www.cjphysiology.org/preprintarticle.asp?id=375111

Ya-Wen Wang and Weiguo Zhang contributed equally to this study, and should be considered as co-first authors.

Introduction

Exosomes are a class of membranous vesicles, measuring 40–150 nm in diameter. These are released into the extracellular matrix from several cell types.^[1],[2],[3] We previously identified exosomes in nipple discharge of breast cancer.^[4] Several studies have reported that microRNAs (miRNAs) extracted from body fluids serve diagnostic purposes in malignant tumors.^[5] Nipple discharge is a relatively common complaint in breast disease. The type of discharge and cytology are unreliable for diagnosis.^[6] Numerous studies have identified potential biomarkers in nipple discharge to assist in the diagnosis of breast cancer.^[7] For example, tumor markers, including carcinoembryonic antigen and CA153, in nipple discharge can facilitate the diagnosis of different breast diseases.^[8] Human nipple discharge, a special body fluid, also contains miRNAs. In our previous study, we identified the differential expression of hsa-miR-4484, kshv-miR-K12-5-5p, hsa-miR-3646, and hsa-miR-4732-5p in nipple discharge between benign tumors and breast cancers.^[9]

miRNAs are stably expressed in body fluids due to the protection conferred by extracellular vesicles and the avoidance of contact with RNase.^{[10],[11],[12]} For example, immune-related miRNAs are stably expressed in exosomes in human breast milk and are resistant to relatively harsh conditions.^[13] Exosomal miRNAs have an important function in regulating cancer progression. Accumulating evidence suggests their potential as biomarkers in cancer diagnosis and prognosis.^[14] However, the presence of RNase in nipple discharge and the potential protective effect of exosomes for miRNAs have not yet been documented in nipple discharge.

In this study, fluorescent TTMAAlPc-RNA compounds were used to confirm the presence of RNase in nipple discharge and colostrum. Exosomes could protect miRNAs from RNase degradation. Exosomes in nipple discharge were more resistant to Triton X-100 treatment than those in colostrum. Our findings lay a theoretical foundation for the future development of exosomal miRNAs in nipple discharge as diagnostic markers for breast cancer.

Materials and Methods

Exosome isolation

Colostrum and nipple discharge samples were collected as described previously.^[4] Colostrum and nipple discharge samples were centrifuged at 2000 ×g in 1.5-ml centrifuge bottles at 4°C for 10 min to remove all fat globules. The supernatant was further centrifuged at 12,000 ×g at 4°C for 30 min to eliminate cells and other debris. The supernatant thus obtained was collected and transferred to 5-ml polycarbonate tubes and centrifuged subsequently at 100,000 ×g at 4°C for 60 min. The exosomal pellet was washed and resuspended in phosphate-buffered saline (PBS) and centrifuged again at 100000 ×g at 4°C for 60 min. Finally, the exosomes were resuspended in PBS and stored at −80°C. Written informed consent was obtained from each patient enrolled in this study. The study protocol and informed consent were approved by the Ethical Committee of Qilu Hospital of Shandong University (approval number: KYLL-2018-096).

RNA isolation

Total RNA was extracted using TRIzol-LS reagent (Ambion) following the manufacturer's protocol. The RNA concentration and purity were confirmed based on the spectrophotometric ratio using absorbance measurements at 260 nm and 280 nm on a Nanodrop 2000 system (Thermo).

Quantitative real-time polymerase chain reaction

The protocol and primers for quantitative real-time polymerase chain reaction (qRT-PCR) have been described previously.^[9] Briefly, 1000 ng of total RNA was used for reverse transcription using the PrimeScript RT reagent Kit with gDNA Eraser (perfect real-time) (TaKaRa) according to the manufacturer's protocol. Then, the FastStart Universal SYBR Green Master (Rox) (Roche) was used to detect and quantify miRNA expression on the StepOne^™ Real-time PCR instrument (Applied Biosystems) following the manufacturer's recommendations. The cycle conditions were as follows: 95°C for 10 min, followed by 40 cycles at 95°C for 10 s, and 58, 60, or 62°C for 30 s, depending on the primers used. The samples were loaded in triplicate and the results of each sample were normalized to the levels of cel-lin-4-5p and cel-miR-2-3p by the 2^−ΔΔCt method.

TTMAAlPc-RNA complex method to measure RNase concentration

RNase concentrations in colostrum and nipple discharge samples were measured by the TTMAAlPc-RNA complex method described by Yang et al.^[15] Briefly, in a 4-ml centrifuge tube, 300 μL of phosphate buffer (pH 7.5, 0.2 mol/L), 30 μL of TTMAAlPc (1 × 10⁻⁴ mol/L), and 11.5 μL of yeast RNA solution (0.8 g/L) (Invitrogen) were added. The RNase A solution (BL543A, Biosharp) or sample for testing was added to a final volume of 3 ml. After the solution was mixed well, the reaction was run in a metal bath at 30°C for 30 min, and subsequently, fluorescence was measured after the reaction system cooled down to room temperature. The excitation and emission wavelengths were set at 610 and 676 nm, respectively, with an emission slit of 6.0 nm. The tube without RNase solution served as a blank control.

The fluorescence intensity of the above system was measured at gradient concentrations of RNase A to draw a calibration curve for RNase. A linear regression equation was obtained as follows: y (fluorescence intensity) = 0.8889 × (RNase concentration) + 6.467, R² = 0.9902. Colostrum or nipple discharge (200 μL) was centrifuged at 12,000 r/min for 8 min, and the supernatant was collected and tested further.

Identification and characterization of exosomes

Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were used to determine the morphology, size, and concentration of exosomes, as described previously.^[4]

Anti-TSG101 (1:1000, Proteintech) and anti-CD81 (1:100, Santa Cruz Biotechnology) primary antibodies were used as exosome protein markers in the western blot analysis.^[4]

Statistical analysis

Statistical analyses were performed using GraphPad Prism 5 (San Diego, CA, USA). Differences between the two groups were analyzed by Student's t-test. A two-tailed P < 0.05 was considered statistically significant.

Results

RNase is present in both colostrum and nipple discharge and degrades exogenous microRNAs

The TTMAAlPc-RNA complex method is a novel test to determine the RNase concentrations.^[15] The fluorescence intensity showed a good linear relationship with the RNase concentration [Figure 1]a. One colostrum specimen and three nipple discharge specimens were tested. The RNase concentration in colostrum was 9.911 mg/ml. RNase concentrations in the three nipple discharge samples were 2.842 mg/ml, 11.560 mg/ml, and 13.798 mg/ml, respectively [Figure 1]b. These results showed that colostrum and nipple discharges were rich in RNase.

Figure 1: RNase concentration measurement by the TTMAAlPc-RNA complex method. (a) The calibration curve for RNase. (b) The RNase concentration in one colostrum and three nipple discharge samples were tested by the TTMAAlPc-RNA complex method. (c and d) The relative expression of exogenous cel-lin-4-5p and cel-miR-2-3p were measured by qRT-PCR at 0, 24, 48, and 72 h in colostrum and nipple discharge samples. qRT-PCR: Quantitative real-time polymerase chain reaction.

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Two exogenous chemically synthesized miRNAs, cel-lin-4-5p and cel-miR-2-3p, were separately added to the colostrum and nipple discharge samples. Expressions of cel-lin-4-5p and cel-miR-2-3p at 0, 24, 48, and 72 h were measured by qRT-PCR analysis. cel-lin-4-5p and cel-miR-2-3p were almost completely degraded at 24, 48, and 72 h in colostrum [Figure 1]c and nipple discharge [Figure 1]d samples. This result indicated that the RNase present in the colostrum and nipple discharge was functional.

hsa-miR-4732-5p, hsa-miR-3646, hsa-miR-4484, and kshv-miR-K12-5-5p are stably expressed in colostrum and nipple discharge

Specimens received from the clinical laboratory were processed immediately or temporarily stored at 4°C and processed within 24 h. In the survey of ten randomly selected patients in Qilu Hospital, the time from collection of nipple discharge samples to their arrival at the specimen processing center ranged from 6 to 42 min, with an average of 23.7 min [Figure 2]a.

Figure 2: Stability of colostrum and nipple discharge-derived exosomal miRNAs. (a) The time from nipple discharge collection to arrival at the specimen processing center of ten randomly chosen samples in Qilu Hospital. (b and c) CT values of miRNAs in colostrum and nipple discharge stored at room temperature for 0, 5, 10, 30, and 60 min. (d and e) CT values of miRNAs in colostrum and nipple discharge at 0 h, 24 h, 48 h, and 72 h at 4°C. miRNAs: microRNAs, CT: Cycle threshold.

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In our previous study, hsa-miR-4732-5p, hsa-miR-3646, hsa-miR-4484, and kshv-miR-K12-5-5p were found to be potential diagnostic markers in nipple discharge samples in breast cancer.^[9] Herein, at room temperature, hsa-miR-4732-5p, hsa-miR-3646, hsa-miR-4484, and kshv-miR-K12-5-5p were found to be stably expressed in colostrum and nipple discharge samples at 0, 5, 10, 30, and 60 min, respectively [Figure 2]b and [Figure 2]c. These miRNAs were also stably expressed in colostrum and nipple discharge samples at 4°C at 0, 24, 48, and 72 h [Figure 2]d and [Figure 2]e. These results suggest that a protective mechanism may exist to prevent miRNAs from degradation by RNase.

Colostrum and nipple discharge derived-exosomes protect hsa-miR-4732-5p, hsa-miR-3646, hsa-miR-4484 and kshv-miR-K12-5-5p from RNase degradation

TEM showed that the isolated extracellular vesicles were approximately 100 nm in diameter and were rounded with a cup-like concavity, which is well-known exosome morphology [Figure 3]a. NTA showed that the median diameter of exosomes was 137.5 nm, and the concentration was 4.5 × 10⁶ particles/mL [Figure 3]b. The exosome marker proteins, CD81, and tumor susceptibility 101 (TSG101) were detected in these exosomes [Figure 3]c.

Figure 3: Identification of exosomes. (a) Extracellular vesicles were isolated by ultracentrifugation and observed by TEM. Scale bar = 500 nm. (b) NTA to detect exosome sizes and concentrations. The median diameter of exosomes was 137.5 nm and the concentration was 4.5 × 10⁶ particles/mL. (c) Western blot results of the exosomal membrane protein, CD81 and the membrane binding protein, tumor susceptibility 101 (TSG101). TEM: Transmission electron microscopy, NTA: Nanoparticle tracking analysis.

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Exosomes isolated from nipple discharge and colostrum were treated as follows: (1) RNase group: RNase A and RNase T1 (100 μg/mL), 37°C, 60 min; (2) Triton X-100 + RNase group: 1% Triton X-100, 37°C, 30 min, and RNase A and RNase T1, 37°C, 60 min, and (3) control group: Untreated. Next, the levels of exosomal hsa-miR-4732-5p, hsa-miR-3646, hsa-miR-4484, and kshv-miR-K12-5-5p were detected [Figure 4]a.

Figure 4: Colostrum- and nipple discharge-derived exosomes protect miRNAs from RNase degradation. (a) Flow chart of degradative treatments for endogenous miRNAs. (b) The relative levels of colostrum exosomal miRNA expression. (c) The relative levels of nipple discharge exosomal miRNAs expression (ns, not significant; *P < 0.05, **P < 0.01). miRNAs: microRNAs.

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In colostrum, compared to the control group, the levels of hsa-miR-4732-5p, hsa-miR-3646, hsa-miR-4484, and kshv-miR-K12-5-5p in the RNase group showed no significant changes (P = 0.1125, 0.2483, 0.0748, and 0.2153, respectively). The levels of hsa-miR-3646, hsa-miR-4484, and kshv-miR-K12-5-5p decreased significantly in the Triton X-100 + RNase group (P = 0.0175, 0.0064, and 0.0018, respectively) compared to the control group, but this trend was not observed for miR-4732-5p (P = 0.2807). The levels of all four miRNAs in the Triton X-100 + RNase group decreased significantly compared to the RNase group (P = 0.0069, 0.0122, 0.042, and 0.0362, respectively) [Figure 4]b.

In nipple discharge, there were no significant differences in the levels of hsa-miR-4732-5p, hsa-miR-3646, hsa-miR-4484, and kshv-miR-K12-5-5p between the RNase and control groups (P = 0.5143, 0.0876, 0.446, and 0.0905, respectively). Interestingly, relative to the control group, the expressions of hsa-miR-4732-5p, hsa-miR-3646, and hsa-miR-4484 did not change significantly (P = 0.542, 0.0834, and 0.736, respectively) in the Triton X-100 + RNase group; only the level of kshv-miR-K12-5-5p decreased (P = 0.0288) in the Triton X-100 + RNase group. In addition, there was no significant difference in the levels of the four miRNAs between the Triton X-100 + RNase and RNase groups (P = 0.9289, 0.9061, 0.4122, and 0.2586, respectively) [Figure 4]c. These results suggested that exosomes in nipple discharge may be more resistant to Triton X-100 treatment than those in the colostrum.

Discussion

Although we have confirmed that both exosomes and miRNAs are present in nipple discharge,^[9],[16] the presence and effectiveness of RNase in nipple discharge remain elusive. To determine RNase concentration in nipple discharge, a novel test based on the TTMAAlPc-RNA system was conducted.^[15] The RNase concentration was measured in colostrum and nipple discharge samples and was between 2.842 and 13.798 mg/ml. To further investigate whether RNase at this concentration range could degrade free miRNAs, two exogenous miRNAs (cel-lin-4-5p and cel-miR-2-3p) were directly added to colostrum and nipple discharge samples. After incubation at 4°C for 24 h, both exogenous miRNAs could hardly be detected by qRT-PCR, indicating that free miRNAs could not resist RNase degradation without exosomal protection.

Samples delivery time for nipple discharge at room temperature in Qilu Hospital was within 60 min, and they were either tested immediately or stored at 4°C and tested within 24 h. Stability experiments were designed and the levels of the four miRNAs (hsa-miR-4732-5p, hsa-miR-3646, hsa-miR-4484, and kshv-miR-K12-5-5p) remained stable at room temperature for 1 h and up to 72 h at 4°C. A similar phenomenon has been observed in human milk and other body fluids.^[13],[17]

The protective effect of exosomes on miRNAs depends on the integrity of the membrane, and some detergents such as Triton X-100 can dissolve lipids, thus increasing membrane permeability.^[18] Studies have shown that the addition of Triton X-100 to exosomes obtained from the serum of osteoclasts and bladder cancer patients can destroy the exosomal membrane structure, leading to the degradation of RNA by RNase.^[19],[20] A similar phenomenon was found in colostrum-derived exosomes in our study. However, the addition of Triton X-100 and two RNases to the nipple discharge-derived exosomes did not significantly decrease the expression of miRNAs, suggesting that Triton X-100 may not destroy the membrane structure of the nipple discharge-derived exosomes. Exosomes in nipple discharge may have stronger membranes since they are more resistant to Triton X-100 treatment than those in the colostrum. Similar results have been reported for human saliva-derived exosomes,^[21] whereby Triton X-100 did not completely destroy exosomal membrane integrity. Kumeda et al. propose that exosomes with core structures consisting of proteins and RNAs may be rigid and resistant to Triton X-100.^[21] These researchers are also interested in the effects of detergents on the membrane integrity of exosomes obtained from various sources. We increased the concentration of Triton X-100 and treatment time and repeated the experiments for several times but Triton X-100 + RNase failed to degrade miRNAs in nipple discharge. This may be explained as follows: colostrum exosomes are mainly secreted by normal ductal lobular epithelial cells, while nipple discharge exosomes are mainly derived from intraductal tumor cells. The two kinds of exosomes may have different membrane structures, and the specific underlying mechanism warrants further studies. Other methods to destroy exosome membranes should be tested in the future. Osteikoetxea et al. found that different extracellular vesicle subpopulations (exosomes, microvesicles, and apoptotic bodies) had different sensitivities to detergent lysis (sodium dodecyl sulfate, Triton X-100, Tween 20, and deoxycholate).^[18] These researchers proposed that differential sensitivity to detergent lysis helps differentiate between exosomes and other extracellular vesicle subpopulations, as well as between vesicular and nonvesicular structures.^[18] Herein, we provide evidence that exosomes from different body fluids or of different pathophysiological states may show differences in resistance to detergent lysis and warrant further investigation.

For the establishment of exosomal miRNAs as biomarkers for the diagnosis of nipple discharge-related breast diseases, several issues need to be addressed. First, differential exosomal miRNA expression profiles should be screened between benign and malignant nipple discharge, as well as breast cell lines, and verified in a large number of clinical samples. Second, we purified high-quality exosomes from nipple discharge by ultracentrifugation. However, there are many other methods to isolate exosomes such as using a precipitation kit or immune magnetic beads. The optimal method of extracting exosomes from nipple discharge is worth examining. Moreover, a regulatory network of exosomal miRNAs should be verified. Finally, long-term follow-up studies are required to confirm the relationship between exosomal miRNA levels and carcinogenesis and the development of breast cancer.

This study has some limitations and further investigations are needed in the future. For example, different concentrations of Triton X-100 and incubation periods should be tested to clarify the half-life of miRNAs and the size and number of exosomes. Triton X-100 is the most used surfactant to test the stability of exosomes, however, other alternatives, such as sodium dodecyl sulfate, Nonidet P-40 as well as sonication, should be evaluated to ensure that the exosomes truly possess protective characteristics. Moreover, differential sensitivity of nipple discharge and colostrum exosomes toward Triton X-100 was observed, and the underlying mechanism should be elucidated in further studies.

Conclusion

In summary, exosomal miRNAs in nipple discharge are stable under degradative conditions. Nipple discharge exosomes were more resistant to Triton X-100 than colostrum exosomes. Future studies are needed to test the protective characteristics of exosomes and investigate the mechanism of differential detergent sensitivity of nipple discharge- and colostrum-derived exosomes. This is expected to facilitate the application of exosomal miRNAs in nipple discharge as potential biomarkers for breast cancer.

Acknowledgments

We thank Prof. Donghui Li from the Cancer Research Center, Medical College of Xiamen University (Xiamen, China) for providing TTMAAlPc-RNA complex, Prof. Yunxue Zhao from the Department of Pharmacology, School of Medicine, Shandong University (Jinan, China) for help in measuring RNase concentration and Yao Liu from the Second Affiliated Hospital of Xi'an Jiaotong University (Xi'an, China) for her help in preparing the manuscript.

Financial support and sponsorship

This work was supported by the National Natural Science Foundation of China (No. 81902698 and 81802406), Shandong Provincial Natural Science Foundation (No. ZR2022MH248 and ZR2020LZL009), the Funding for New Clinical and Practical Techniques of Qilu Hospital of Shandong University (No. 2019-1), and Special Funds for Scientific Research on Breast Diseases of Shandong Medical Association (No. YXH2021ZX058).

Conflicts of interest

There are no conflicts of interest.

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