Skip to main content
NCBI home page
PLOS One logoLink to PLOS One
. 2022 Sep 22;17(9):e0274919. doi: 10.1371/journal.pone.0274919

MicroRNA-206 suppresses growth and metastasis of breast cancer stem cells via blocking EVI-1-mediated CALR expression

Dapeng Sun 1,*, Chenguang Li 1, Fengxiang Zhang 2,*
Editor: Ning Wei3
PMCID: PMC9498949  PMID: 36136972

Abstract

Aim to investigate the effect of miR-206 on the growth and metastasis of breast cancer stem cells and clarify the precise mechanism of miR-206 on EVI-1-mediated CALR expression in driving malignant phenotype. Our results showed that miR-206 mimics suppressed CALR expression, inhibited the proliferation and metastasis ability of breast cancer stem cells and finally induced cellular apoptosis. Over-expression of CALR could effectively attenuate the cytotoxic effect of miR-206. Further studies demonstrated that EVI-1 could be served as a key regulator of miR206-mediated CALR expression. Elevation of EVI-1 can reverse the function of miR-206 on induction of CALR.

Introduction

Breast cancer is the most common malignant tumor in female cancers. Currently, the main treatment methods are surgery, radiotherapy, chemotherapy and biological therapy. However, the development of drug resistant is an obstacle to successfully treatment for breast cancer. Recently studies have confirmed that there are a small subgroup of breast cancer stem cells in breast cancer tissues [1, 2], which not only have high tumorigenicity and high invasion and metastasis, but also have the characteristics of resistance to chemotherapy and radiotherapy. They play important roles in the development, metastasis and resistance in breast cancer. Therefore, discovering novel biological markers of breast cancer stem cells, developing corresponding molecular targeted drugs, and carrying out multi-sites targeted drug therapy will contribute to effective treatment for breast cancer [3, 4].

Calreticulin (CALR), a multifunctional calcium-binding molecular chaperone, mainly locate in cellular endoplasmic reticulum (ER). The abnormal expression of CALR occurs in different types of tumors [5]. Previous studies showed that calreticulin could significantly affect cell proliferation, invasion, induction of apoptosis and angiogenesis in glioma tissues [6]. Studies have confirmed that calreticulin expression was up-regulated and mainly localized in the cytoplasm in breast cancer MDA-MB-231 stem cells. The expression level of calreticulin in highly invasive MDA-MB-231 stem cells was significantly higher than that of weakly invasive MCF-7 stem cells [7].

MicroRNAs, a class of non-coding RNA molecules of 19–25 nucleotides, are widely involved in the regulation of development, proliferation, differentiation and apoptosis [8–10]. Recently, microRNAs have rapidly developed into important molecular markers of tumors and other diseases [11, 12]. MiR-206 is a typical multifunctional microRNA that located in the non-coding gene Bic and expressed in a variety of cancer cells. It has been shown that miR-206 plays an important role in the process of immune response, inflammation, tumorigenesis and so on [13, 14]. There are also reported that the expression of miR-206 was down-regulated in estrogen receptor-alpha positive breast cancer MDA-MB-231 stem cells [15], and the overexpression of miR-206 in breast cancer MDA-MB-231 stem cells can effectively inhibit cells growth. Although these studies have shown that miR-206 plays an important role in the development and progression of breast cancer stem cells, it is still unclear how it regulates the growth of breast cancer stem cells.

In this study, we investigate the potential mechanism of miR-206 affecting the biological activity of breast cancer stem cells by interfering with the expression of CALR, and then providing novel targets and new strategy for the prevention and treatment of breast cancer.

Materials and methods

Chemicals and agents

miR-206 mimics, Ad-EVI-1, and Ad-CALR (Ambion,Tokyo, Japan); BCA Protein Assay Kit (Catalog number: 23225, Pierce Chemical, Rockford, USA); anti-EVI-1(463–477), anti-E-cadherin (SRP6426), anti-CALR (AV30114), anti-N-cadherin (05–915) were purchased from Sigma-Aldrich, USA; the Annexin V-FITC Apoptosis Detection Kit (Beyotime Institute of Biotechnology, Jiangsu, China); Total RNA was isolated from breast CSCs with RNAisoPlus (Takara Biotechnology Co, Ltd., Dalian, China). All cell culture medium and fetal bovine serum were purchased from Gibco (Thermo Fisher scientific, USA).

Cell lines and cell culture

Human breast cancer cell line MDA-MB-231 was purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). MDA-MB-231 cells were digested by trypsin and harvested. The cells were labeled with anti-human CD44-fluorescein isothiocyanate (FITC), anti-human CD24-phycoerythrin and anti-human ESA-PerCP-Cy5.5-A antibodies for 20 minutes. Breast cancer stem cells were sorted and cultured in DMEM/F12 (1:1) medium with 20 μg/L basic fibroblast growth factor, 10 μg/L epidermal growth factor and 2% B27, incubated at 37°C, 5% CO2.

Transfection experiments

MiR-206 mimics, Ad-EVI-1 and Ad-CALR were transfected into human MDA-MB-231 breast cancer cells. After that, the cells were digested by trypsin and harvested. Labeled with anti-human CD44-fluorescein isothiocyanate (FITC), anti-human CD24-phycoerythrin and anti-human ESA-PerCP-Cy5.5-A antibodies for 20 minutes, breast cancer stem cells were sorted and cultured in DMEM/F12 (1:1) medium with 20 ug/L basic fibroblast growth factor, 10 ug/L epidermal growth factor and 2% B27, incubated at 37°C, 5% CO2. Breast cancer stem cells were inoculated into 6-well plates, cultured to 50% fusion, transfected with Lipofectamine 2000, and cultured in 10% fetal bovine serum for 24 hours. Then a new medium containing 100 U/mL penicillin and 100ng/mL streptomycin was replaced. Transfection efficiency was confirmed by quantitative RT-PCR.

MTT assay

Breast cancer stem cells (2x103) were seeded in 96-well plates, then transfected with 50 nM miR-206 and/or Ad-CALR constructs and incubated for 48 hours. The supernatant was removed, and an MTT solution was added to each well and cultured at 37°C for additional 3 hours. The culture solution was aspirated, DMSO was added to each well and shaken for 10 min on a constant temperature shaker and incubated at room temperature for 20 min. The OD value was measured by microplate reader at 570 nm.

Flow cytometry analysis

After transfection of 50 nM miR-206 or/and Ad-CALR for 48 hours, the cells were washed twice with cold PBS and resuspended in 1 x Binding Buffer. Take 195 μl of cell suspension in a 5 ml flow tube, add 5 μl of Annexin V-FITC, mix gently and mix for 3 min, then add 10μl or 20 μg/ml of PI solution, shake, and let stand for 15 min at room temperature in the dark. The tube was added with 400 μl of 1 x Binding Buffer. The apoptotic cells were astained and analyzed by flow cytometry.

Transwell assay detects the invasion of breast cancer stem cells

Breast cancer stem cells were transfected with 50 nM miR-206 or/and Ad-CALR constructs and incubated for 48 hours. A single cell suspension was prepared and the cell concentration was adjusted to 2×105. Matrigel was used to cover the PVP membrane between the upper and lower rooms of Transwell and treated at 37°C for 30 minutes. 100 μl of cells were added to the upper chamber, 500μl of serum-containing medium was added to the lower chamber. Each group was divided into three compound pores. After 24 hours of incubation in CO2 incubator, the cells were removed and fixed with 95% ethanol for 10 minutes. Conventional 0.1% crystal violet staining, five visual fields were randomly taken under the microscope, up, down, left, right and center. The number of perforating cells was counted. The average number of each visual field was taken to indicate the invasiveness of tumor cells.

Western blot analysis

As our previously described, equal amounts of protein were loaded onto an 8% SDS-PAGE gel and transferred to a polyvinylidene fluoride membrane.5% skimmed milk was sealed for 2 hours and then with anti-EVI-1 (1: 1,000), anti-CALR (1:500), anti-E-cadherin (1: 1,000) or anti-N-cadherin (1: 1,000) incubate together. After rinsed by TBST buffer, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody for 1 hour at a concentration of 1:5000. Finally, the density of immunoblot was quantified by image J software.

Real-time quantitative PCR assay

Total RNA was isolated from breast cancer stem cells with RNA Plus according to the manufacturer’s instructions. cDNA for microRNA and gene expression analyses was prepared using the miScript II RT kit and RT2 the first strand kit, respectively. The qRT-PCR was performed in triplicate using the miScript SYBR Green PCR kit and the Quantitech SYBR green kit on a StepOnePlus real time PCR system. Relative changes in expression were calculated using the 2-ΔΔCt method, where ΔCt is the difference in threshold cycles for the target gene and reference (ACTB), and ΔΔCt is the difference between the ΔCts of the treated sample and control or calibrator. Thus, the expression levels were reported as fold changes relative to the calibrator. The primers as follow: CALR- F 5′-TGG TCC TGG TCC TGA TGT CG-3′ and CALR-R 5′-CTC TAC AGC TCG TCC TTG-3′; ACTN-F AGG TCA TCA CCA TCG GCA ACG A, ACTN-R GCT GTT GTA GGT GGT CTC GTG A.

Statistical methods

All data were analyzed by one-way ANOVA method using SPSS 13.0 software. P < 0.05 indicates that the difference has statistical significance.

Results

Effect of miR-206 on the expression of CALR in breast cancer stem cells

To investigate the effect of miR-206 on CALR expression in human breast cancer stem cell, the breast cancer stem cells were firstly sorted and collected as CD44+ and CD24+ positive population and then were transfected with 30, 50 and 70nM of miR-206 for 48 hours. As seen in Fig 1, miR-206 mimics significantly suppressed the protein and mRNA expression level of CALR in breast cancer stem cells in a dose-dependent manner. Treatment of miR-206 (50nM) resulted in approximately 50% reduction of CALR expression.

Fig 1. Effect of miR-206 on CALR expression in breast cancer stem cells.

Fig 1

Open in a new tab

Human breast cancer stem cells were treated with various concentrations of miR-206 mimics (0, 30, 50, 70 nM) for 48 h. CALR protein expression (A&B) and mRNA expression (C) level were measured by Western blot and real-time quantitative PCR, respectively; Human breast cancer stem cells were treated with 50 nM miR-206 mimics for 0, 24, 48 and 72h. CALR protein expression (D&E) and mRNA expression level were determined by Western blot and real-time quantitative PCR, respectively. All results are expressed as the mean ± S.D. from three independent experiments. *P < 0.05, **P < 0.01 vs. control group.

Furthermore, to determine the time-course inhibitory effect of miR-206 on CALR expression, human breast cancer stem cells were transfected with miR-206 mimics (50nM) at different time point (0, 24, 48 and 74 hours), and the mRNA and protein expression level of CALR was determined by RT-PCR and western blot analysis, respectively. As shown in Fig 1D, we observed that the significantly suppression effect on CALR expression in breast cancer stem cells when the cells were exposed to 50nM of miR-206 mimics for 48 or 72 hours. (Fig 1D–1F).

Overexpression of CALR attenuate the cytotoxic effect of miR-206 in breast cancer stem cells

To verify that CALR can be acted as the target of miR-206, the ad-CALR was designed and transfected into breast cancer stem cells for 48 hours. As shown in Fig 2A & 2B, the expression of CALR was significantly elevated in ad-CALR alone group, and miR-206 alone significantly reduced CALR expression. While the co-transfection of ad-CALR and miR-206, the overexpression of CALR could attenuate the inhibitory effect of miR-206 on CALR expression (P <0.05). The effect of ad-CALR and/or miR206 on the growth of breast cancer stem cells was determined by MTT assay. As seen in Fig 2C, miR-206 mimics alone treatment for 48 hours resulted in 52.96% suppressing cell growth, which significant inhibited the proliferation of breast cancer stem cells as compared to the control group (P<0.05). Meanwhile, we observed that the transfection with Ad-CALR promoted the growth of cells. The breast cancer stem cells were transfected with Ad-CALR in combination with miR-206 mimics for 48 hours, the inhibitory effect of miR-206 was reversed by overexpression of CALR.

Fig 2. Overexpression of CALR attenuate the cytotoxic effect of miR-206 on growth of breast cancer stem cells.

Fig 2

Open in a new tab

Human breast cancer stem cells were treated by miR-206 and/or Ad-CALR for 48 h, then the cells were harvested and analyzed. (A&B) CALR protein expression were determined by immunoblot analysis; (C) Human breast cancer stem cells were transfected with miR-206 and/or Ad-CALR for 72 h, the cell growth was determined by MTT assay; (D&E) Effect of CALR overexpression attenuate the apoptosis effect of miR-206 in breast cancer stem cells. Human breast cancer stem cells were treated by miR-206 and/or Ad-CALR for 48 h, the early and late apoptotic cells were measured by PI-Annexin V, *P < 0.05, **P < 0.01 vs. control group.

The results showed that miR-206 can significantly inhibit the growth of breast cancer stem cells, but the effect of miR-206 on the growth of breast cancer stem cells was significantly attenuated by overexpression of CALR (Fig 2C). In addition to the MTT assay, the effect of miR-206 on inducing cellular apoptosis was measured by PI-AnnexinV staining assay. As seen in Fig 2D & 2E, the cells were treated by miR-206 alone for 48 hours, the late apoptotic cells were increased to 21.96%. Meanwhile, we found that overexpression of CALR reduced the cells undergoing apoptosis (6.96%). What is more, the apoptotic cells induced by miR206 were significantly deceased when the cells were co-transfected with Ad-CALR. Thus, overexpression of CALR could effectively attenuate the cytotoxic effect of miR-206 in breast cancer stem cells.

Effect of miR-206 on CALR-mediated metastasis of human breast cancer stem cells

Next, the effect of miR-206 mimics and/or ad-CALR on the ability of invasion and migration was measured by using transwell assay. As seen in Fig 3A, the breast cancer stem cells were transfected with miR206 alone for 48 hours, the invasive cells were decreased 31.9%, as compared to the control group (P<0.05). Overexpression of CALR enhanced the ability of breast cancer stem cells, and the invasive cells were increased to 127.7%, (p<0.05). What is more, the co-transfection of Ad-CALR significantly reduced the suppression of miR206 on invasive breast cancer stem cells. Our results suggested that miR-206 could significantly inhibit the invasion ability of breast cancer stem cells, whereas the ability of miR-206 to inhibit the invasion of breast cancer stem cells is reduced by overexpression of CALR. (Fig 3).

Fig 3. Effect of miR-206 on CALR-mediated metastasis of human breast cancer stem cells.

Fig 3

Open in a new tab

Cells were treated with miR-206 mimics, miR-206 mimics+Ad-CALR and Ad-CALR for 48 h, respectively. (A) invasive breast cancer stem cells were counted; (B&D), CALR protein expression was measured by Western blotting analysis; C, CALR gene was measured by real-time quantitative PCR. All results are expressed as the mean ± S.D. from three independent experiments, *P < 0.05, **P < 0.01; #P<0.05, ##P<0.01 vs. control group.

EMT (epithelial-mesenchymal transition) is essential for initiation of metastasis in tumor progression. Given the effect of miR206/CALR on cell invasion, expression level of key regulators in EMT signaling pathway was determined by western blot analysis, including E-cadherin and N-cadherin. As seen in Fig 3B, overexpression of CALR enhanced the E-cadherin and reduced N-cadherin expression. miR-206 alone resulted in a decrease of E-cadherin and increase of N-cadherin. Co-transfection of Ad-CALR could partially reduce the biological effect of miR206 on EMT pathway. This result is consistent to our transwell assay. In addition, our results provide the evidences that miR-206 can regulate the expression of EMT-related genes by enhancing the expression of CALR.

EVI-1 is associated with miR206/CALR signaling in human breast cancer

To clarify the mechanism of miR-206 on CALR expression, the effect of EVI-1 on CALR expression was investigated. As shown in Fig 4A & 4B, CALR expression in breast cancer stem cells was significantly increased in Ad-EVI-1 group, as compared to the control group; while knockdown of EVI-1 (si-EVI-1) effectively reduced CALR expression in breast cancer stem cells (P<0.05). There is a positive correlation of CALR and EVI-1 in breast cancer stem cells.

Fig 4. EVI-1 is involved in miR-206/CALR in human breast cancer stem cells.

Fig 4

Open in a new tab

(A&B) breast cancer stem cells were transfected with Ad-EVI-1 or si-EVI-1 for 48 h, the EVI-1 expression was determined by immunoblot analysis; (C&D) Human breast stem cell were treated with miR-206 mimics, miR-206 mimics+Ad-EVI-1 and Ad-EVI-1 for 48 h. EVI-1 and CALR expression were measured by Western blotting analysis; (E), the mRNA level of EVI-1 and CALR were measured by real-time quantitative PCR; (F), Schematic of MiR/206-EVI-1/CALR pathway in human breast cancer. All results are expressed as the mean ± S.D. from three independent experiments, *P < 0.05, **P < 0.01; #P<0.05, ##P<0.01 vs. control group.

To further confirm the function of EVI-1 in miR206-mediated CALR expression, the effect of Ad-EVI and/or miR-206 on EVI-1 and CALR expression were determined by western blot analysis and RT-PCR, respectively. As the results shown in Fig 4C & 4D, EVI-1 and CALR expression in breast cancer stem cells were significantly suppressed in the miR-206 mimics group, as compared to the control group (P<0.05). The expressions of EVI-1 and CALR were significantly enhanced in miR-206 mimics plus Ad-EVI-1 group and Ad-EVI-1 alone group (P<0.05). These results indicated that the inhibitory effect of miR-206 on the expression of CALR may be reversed the overexpression of EVI-1.

Discussion

miR-206 has been detected in various types of tumor cells, including lung cancer, gastric cancer, breast cancer and colon cancer. Functionally, MiR-206 can influence fate of tumor cells by regulating the expression levels of different signaling pathways in tumor cells, and then manipulate the formation of tumors [16, 17]. Breast cancer stem cells not only retain the basic characteristics of normal cells, but also have strong self-renewal ability and tumorigenicity. This feature may be one of the main reason of recurrence and metastasis of breast cancer, but the effect and precise mechanism of miR206 on proliferation and metastasis of breast cancer stem cells is unknown.

The expression level of CALR is positively correlated with tumor size and status of metastasis. Thus, CALR can be used as an auxiliary biomarker to guide diagnosis, treatment and prognosis [18]. Herein, we discovered that miR-206 mimics inhibited CALR expression in a time- and dose-dependent manner. RT-PCR results confirmed that miR-206 mimics significantly suppressed CALR mRNA. Our results suggest that enhancing the expression of miR-206 can significantly inhibit CALR expression in breast cancer stem cells.

Further studies demonstrated that miR-206 mimics significantly induced apoptosis and suppressed the proliferation and invasion ability of breast stem cells. Interestingly, overexpression of CALR could attenuate the effect of miR-206 mimics. However, there was no significant difference between the Ad-CALR alone and control group. This result suggested that miR-206 play an important role in growth of breast cancer stem cells, and closely related to CALR expression.

EMT play an important role in the invasion and metastasis of malignant tumors, and main change in EMT in tumor cells is a decrease in the expression of the epithelial marker E-cadherin and an increase in the expression of the interstitial marker N-cadherin [19–21]. Overexpression of N-cadherin is closely related to the invasion and metastasis of a variety of epithelial-derived malignant tumors. When N-cadherin is over-expressed, the structure of tissue cells becomes loose, the adhesion between cells decreases, and then cells Invasion and transfer [22, 23]. Herein, we observed that miR-206 could significantly enhance the expression of E-cadherin and decrease the expression of N-cadherin. What is more, this effect was reversed when the expression of CALR was upregulated. Our results indicated that miR-206 altered regulation of E-cadherin and N-cadherin through CALR-dependent manner in breast cancer stem cells.

Recently, researchers found that the expression of EVI-1 (ecotropic virus integration-1) is up-regulated in metastatic breast cancer stem cells. High expression of EVI-1 can lead to poor prognosis in breast cancer patients with estrogen receptor negative [24–26], suggesting that transcription factor EVI-1 may play a carcinogenic role by promoting the growth, migration and invasion of breast cancer stem cells. Our study demonstrated that enhancing the expression of EVI-1 could significantly enhance the expression of CALR in breast cancer stem cells, and there was a significant positive correlation between CALR and EVI-1. Further study indicated that high-level of miR-206 could reduce the expression of EVI-1 and inhibit the activity of CALR, but when the expression of EVI-1 was enhanced, the effect of miR-206 on inhibiting CALR activity was significantly reduced. These results suggested that miR-206 was likely to regulate the activity of CALR by reducing the expression of EVI-1.

Taken together, miR-206 suppresses the growth and metastasis ability of breast cancer stem cells via blocking CALR expression. Further studies demonstrated that regulation of CALR by miR-206 may be realized by affecting the expression of EVI-1. Our result not only clarified the mechanism of miR-206 regulating CALR expression in breast cancer stem cells, but also provided a novel target for the prevention and treatment of breast cancer.

Supporting information

S1 File. Raw images for western blots.

(PDF)

Click here for additional data file. (144.1KB, pdf)

Acknowledgments

We gratefully thank the staff in the Department of Oncology, The First Affiliated Hospital of Jinzhou Medical University who reviewed and made comments on our manuscript.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This study was supported by Liaoning Provincial Natural Science Foundation (20180550601&2019-ZD-0833); Liaoning Provincial Eduication Foundation (JYTCZR2020051). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  • 1.Wendling CA. Evolution of the Perception of the Relationship Between Doctor and Breast Cancer Patient. Adv Mind Body Med. 2016;30(1):28–31. Epub 2016/02/16. . [PubMed] [Google Scholar]
  • 2.Butti R, Gunasekaran VP, Kumar TVS, Banerjee P, Kundu GC. Breast cancer stem cells: Biology and therapeutic implications. Int J Biochem Cell Biol. 2019;107:38–52. Epub 2018/12/12. doi: 10.1016/j.biocel.2018.12.001 . [DOI] [PubMed] [Google Scholar]
  • 3.Villar Balboa I, Regi Bosque M. [Breast cancer in a male patient]. Semergen. 2016;42(7):e110–e2. Epub 2016/03/05. doi: 10.1016/j.semerg.2015.12.018 . [DOI] [PubMed] [Google Scholar]
  • 4.Xu D, Xu H, Ren Y, Liu C, Wang X, Zhang H, et al. Cancer stem cell-related gene periostin: a novel prognostic marker for breast cancer. PLoS One. 2012;7(10):e46670. Epub 2012/10/12. doi: 10.1371/journal.pone.0046670 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Lu YC, Chen CN, Wang B, Hsu WM, Chen ST, Chang KJ, et al. Changes in tumor growth and metastatic capacities of J82 human bladder cancer cells suppressed by down-regulation of calreticulin expression. Am J Pathol. 2011;179(3):1425–33. Epub 2011/07/05. doi: 10.1016/j.ajpath.2011.05.015 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Liu XL, Zhao D, Sun DP, Wang Y, Li Y, Qiu FQ, et al. Adenovirus-mediated delivery of CALR and MAGE-A3 inhibits invasion and angiogenesis of glioblastoma cell line U87. J Exp Clin Cancer Res. 2012;31:8. Epub 2012/02/02. doi: 10.1186/1756-9966-31-8 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Eric-Nikolic A, Milovanovic Z, Sanchez D, Pekarikova A, Dzodic R, Matic IZ, et al. Overexpression of calreticulin in malignant and benign breast tumors: relationship with humoral immunity. Oncology. 2012;82(1):48–55. Epub 2012/02/09. doi: 10.1159/000335267 . [DOI] [PubMed] [Google Scholar]
  • 8.Kwak PB, Iwasaki S, Tomari Y. The microRNA pathway and cancer. Cancer Sci. 2010;101(11):2309–15. Epub 2010/08/24. doi: 10.1111/j.1349-7006.2010.01683.x . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Rupaimoole R, Slack FJ. MicroRNA therapeutics: towards a new era for the management of cancer and other diseases. Nat Rev Drug Discov. 2017;16(3):203–22. Epub 2017/02/18. doi: 10.1038/nrd.2016.246 . [DOI] [PubMed] [Google Scholar]
  • 10.Pasquinelli AE. MicroRNAs and their targets: recognition, regulation and an emerging reciprocal relationship. Nat Rev Genet. 2012;13(4):271–82. Epub 2012/03/14. doi: 10.1038/nrg3162 . [DOI] [PubMed] [Google Scholar]
  • 11.Wu X, Somlo G, Yu Y, Palomares MR, Li AX, Zhou W, et al. De novo sequencing of circulating miRNAs identifies novel markers predicting clinical outcome of locally advanced breast cancer. J Transl Med. 2012;10:42. Epub 2012/03/10. doi: 10.1186/1479-5876-10-42 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Halvorsen AR, Helland A, Gromov P, Wielenga VT, Talman MM, Brunner N, et al. Profiling of microRNAs in tumor interstitial fluid of breast tumors—a novel resource to identify biomarkers for prognostic classification and detection of cancer. Mol Oncol. 2017;11(2):220–34. Epub 2017/02/02. doi: 10.1002/1878-0261.12025 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Liu F, Zhao X, Qian Y, Zhang J, Zhang Y, Yin R. MiR-206 inhibits Head and neck squamous cell carcinoma cell progression by targeting HDAC6 via PTEN/AKT/mTOR pathway. Biomed Pharmacother. 2017;96:229–37. Epub 2017/10/11. doi: 10.1016/j.biopha.2017.08.145 . [DOI] [PubMed] [Google Scholar]
  • 14.Yin K, Yin W, Wang Y, Zhou L, Liu Y, Yang G, et al. MiR-206 suppresses epithelial mesenchymal transition by targeting TGF-beta signaling in estrogen receptor positive breast cancer cells. Oncotarget. 2016;7(17):24537–48. Epub 2016/03/26. doi: 10.18632/oncotarget.8233 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Mitchelson KR, Qin WY. Roles of the canonical myomiRs miR-1, -133 and -206 in cell development and disease. World J Biol Chem. 2015;6(3):162–208. Epub 2015/09/01. doi: 10.4331/wjbc.v6.i3.162 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Park YR, Seo SY, Kim SL, Zhu SM, Chun S, Oh JM, et al. MiRNA-206 suppresses PGE2-induced colorectal cancer cell proliferation, migration, and invasion by targetting TM4SF1. Biosci Rep. 2018;38(5). Epub 2018/08/24. doi: 10.1042/BSR20180664 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Chen QY, Jiao DM, Wu YQ, Chen J, Wang J, Tang XL, et al. MiR-206 inhibits HGF-induced epithelial-mesenchymal transition and angiogenesis in non-small cell lung cancer via c-Met /PI3k/Akt/mTOR pathway. Oncotarget. 2016;7(14):18247–61. Epub 2016/02/27. doi: 10.18632/oncotarget.7570 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Lee HH, Lim CA, Cheong YT, Singh M, Gam LH. Comparison of protein expression profiles of different stages of lymph nodes metastasis in breast cancer. Int J Biol Sci. 2012;8(3):353–62. Epub 2012/03/07. doi: 10.7150/ijbs.3157 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.De Craene B, Berx G. Regulatory networks defining EMT during cancer initiation and progression. Nat Rev Cancer. 2013;13(2):97–110. Epub 2013/01/25. doi: 10.1038/nrc3447 . [DOI] [PubMed] [Google Scholar]
  • 20.Kotiyal S, Bhattacharya S. Breast cancer stem cells, EMT and therapeutic targets. Biochem Biophys Res Commun. 2014;453(1):112–6. Epub 2014/09/28. doi: 10.1016/j.bbrc.2014.09.069 . [DOI] [PubMed] [Google Scholar]
  • 21.Angadi PV, Patil PV, Angadi V, Mane D, Shekar S, Hallikerimath S, et al. Immunoexpression of Epithelial Mesenchymal Transition Proteins E-Cadherin, beta-Catenin, and N-Cadherin in Oral Squamous Cell Carcinoma. Int J Surg Pathol. 2016;24(8):696–703. Epub 2016/11/04. doi: 10.1177/1066896916654763 . [DOI] [PubMed] [Google Scholar]
  • 22.Liu YA, Liang BY, Guan Y, You J, Zhu L, Chen XP, et al. Loss of N-cadherin is associated with loss of E-cadherin expression and poor outcomes of liver resection in hepatocellular carcinoma. J Surg Res. 2015;194(1):167–76. Epub 2014/12/03. doi: 10.1016/j.jss.2014.09.031 . [DOI] [PubMed] [Google Scholar]
  • 23.Bhandari A, Zheng C, Sindan N, Sindan N, Quan R, Xia E, et al. COPB2 is up-regulated in breast cancer and plays a vital role in the metastasis via N-cadherin and Vimentin. J Cell Mol Med. 2019;23(8):5235–45. Epub 2019/05/24. doi: 10.1111/jcmm.14398 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Yuan X, Wang X, Bi K, Jiang G. The role of EVI-1 in normal hematopoiesis and myeloid malignancies (Review). Int J Oncol. 2015;47(6):2028–36. Epub 2015/10/27. doi: 10.3892/ijo.2015.3207 . [DOI] [PubMed] [Google Scholar]
  • 25.Patel JB, Appaiah HN, Burnett RM, Bhat-Nakshatri P, Wang G, Mehta R, et al. Control of EVI-1 oncogene expression in metastatic breast cancer cells through microRNA miR-22. Oncogene. 2011;30(11):1290–301. Epub 2010/11/09. doi: 10.1038/onc.2010.510 . [DOI] [PubMed] [Google Scholar]
  • 26.Wu L, Wang T, He D, Li X, Jiang Y. miR-1 inhibits the proliferation of breast cancer stem cells by targeting EVI-1. Onco Targets Ther. 2018;11:8773–81. Epub 2018/12/26. doi: 10.2147/OTT.S188836 . [DOI] [PMC free article] [PubMed] [Google Scholar]

Decision Letter 0

Ning Wei

31 May 2022

PONE-D-22-08405MicroRNA-206 suppresses growth and metastasis of breast cancer stem cells via blocking EVI-1-mediated CALR expressionPLOS ONE

Dear Dr. zhang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. According to all reviewers' comments, please address the concerns. The quality of this manuscript will be improved.

Please submit your revised manuscript by Jul 15 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Ning Wei

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

“This study was supported by Liaoning Provincial Natural Science 11 Foundation (20180550601); Liaoning Provincial Natural Science Foundation(2019- ZD-0833).”

We note that you have provided additional information within the Acknowledgements Section that is not currently declared in your Funding Statement. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

4. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

5. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

6. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

Additional Editor Comments:

According to all reviewers' comments, please address the concerns. The quality of this manuscript will be improved.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this paper, the authors found that miR-206 mimics suppressed CALR expression, inhibited the proliferation and metastasis ability of breast cancer stem cells and finally induced cellular apoptosis. They further showed that this was correlated with suppression of EVI-1 signaling. The study was supported some solid data. However, few issues need to be addressed.

1. Please clarify how or where did you get the stem cells?

2. The materials and methods part is completely in a mess, please rewrite this part clearly.

3. In Fig 1B, how did the authors qualify the protein expression, please clarify in the methods part.

4. As showed in Fig 1D and E, the suppression effect was stronger at 72 hours instead of 48 hours, please check this result.

5. In Fig 2C and D, why the MTT assay did not inconsistent with flow analysis in Ad-CALR and Ad-CALR + miR-206 mimics group?

6. Could the authors provide representative images of transwell assay?

7. Many grammar mistakes and errors were noted. It is suggested that authors seek for professional editing service to improve the quality of language and the organization of the manuscript.

Reviewer #2: On the whole, it is a valuable and meaningful study, and the design in this study is logical. However, some points should be explained as following before publication.

1. Its research significance and potential application value should be discussed in the discussion

2. How to prove whether the transfection of MiR-206 mimics is successful?

3. What is the function of E-cadherin and N-cadherin in tumor progression? Why CALR exerted different effect on E-cadherin and N-cadherin?

4. Why Ad-CALR can not reverse the effect of MiR-206 on E-cadherin and N-cadherin?

5. Did the Ad-EVI-1 treatment affect the effect of miR-206 mimics on cytotoxic effect, E-cadherin and N-cadherin?

6. Please explained the principle and mechanism of miR-206 mimics, Ad-CALR and Ad-EVI-1 transfected into cells, what is different with plasmid transfection?

Reviewer #3: In this manuscript, Dapeng and co-authors describe the effect of miRNA-206 on the growth and migration of breast cancer cells and explore the underlying mechanism. Defining the role of miRNA-206 in breast cancer progression is useful to develop novel relevant prevention and/or therapy strategy . The authors did some work to validate their proposal. However, this study is not logically clear and some data is not convincing and need more detail work relating the underlying mechanism, particularly in relation to the relationship between miR-206, CALR and EVI-1 in their engaged signaling pathway.

Major comments

1. MM doesn’t clarify the origin of breast cancer stem cell, and more importantly these used stem cells should be fully/extensively characterized prior to functional assay, such as more stem cell markers besides CD44+/ CD24- should be employed to validate stem cells identity.

2. For the stem cells invasion/migration assay, it’s worth to include some representative images, that will be more visualized and convincing.

3. The final schematic is not consistent with the conclusion drawn in the text.

4. It is highly recommended to carefully go over the MM part to sort out some obvious incorrect description such as “transfected with 5ml Lipo 200”, repeated description, and incomplete method description.

5. Please include all the qPCR primers sequence used in this study.

6. The EVI-1 background introduction should be incorporated.

Minor comments

a. The cat # of Reagents/Cells/Instrument used in this study is missing.

b. Please number all the rows of the text part, which will be much easier to be located during the reviewing process.

c. The ms should be further polished regarding its writing in a professional way and tense using.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2022 Sep 22;17(9):e0274919. doi: 10.1371/journal.pone.0274919.r002

Author response to Decision Letter 0

27 Aug 2022

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response: Dear Editor, according to your suggestion, the title page was updated.

2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

Response: Dear Editor, according to your suggestion, the funding information was added on page 10.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

“This study was supported by Liaoning Provincial Natural Science 11 Foundation (20180550601); Liaoning Provincial Natural Science Foundation(2019- ZD-0833).”

We note that you have provided additional information within the Acknowledgements Section that is not currently declared in your Funding Statement. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Response: Dear Editor, according to your suggestion, the acknowledgement was corrected.

4. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

Response: Dear Editor, thank you for your suggestion! The supporting information files were uploaded, include the western blot images.

5. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

Response: Dear Editor, according to you comment, the ORCID (0000-0002-1713-4035) was provided.

6. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

Response: Dear Editor, the western blot images were uploaded.

Point-to-point Response to reviewer

Reviewer #1: In this paper, the authors found that miR-206 mimics suppressed CALR expression, inhibited the proliferation and metastasis ability of breast cancer stem cells and finally induced cellular apoptosis. They further showed that this was correlated with suppression of EVI-1 signaling. The study was supported some solid data. However, few issues need to be addressed.

1. Please clarify how or where did you get the stem cells?

Response: Dear Reviewer#1, thank you for your concern. As described in the method 2.2, MDA-MB-231 cells were digested by trypsin and harvested. The cells were labeled with anti-human CD44-fluorescein isothiocyanate (FITC), anti-human CD24-phycoerythrin and anti-human ESA-PerCP-Cy5.5-A antibodies for 20 minutes. Breast cancer stem cells were cultured in DMEM/F12 (1:1) medium with 20 µg/L basic fibroblast growth factor, 10 µg/L epidermal growth factor and 2% B27, incubated at 37°C, 5% CO2. (on page 4, line 112).

2. The materials and methods part is completely in a mess, please rewrite this part clearly.

Response: Reviewer#1, thank you for your suggestion! As seen on page3 line 94, We carefully revised and edited the part of method and made it clear.

3. In Fig 1B, how did the authors qualify the protein expression, please clarify in the methods part.

Response: thank you for your suggestion! The quantification of protein expression was performed by image J, the sentence was inserted into the part of method (on page 5, line 180).

4. As showed in Fig 1D and E, the suppression effect was stronger at 72 hours instead of 48 hours, please check this result.

Response: Thank you for your comment! The effect of miRNA206 on CALR expression was quantified by image J software, and an updated image was shown in Fig.1D. As seen, the stronger effect of miR-206 was observed at 72 hours.

5. In Fig 2C and D, why the MTT assay did not inconsistent with flow analysis in Ad-CALR and Ad-CALR + miR-206 mimics group?

Response: Dear Reviewer#1, we have double checked the results, the cell viability means the live breast cancer stem cells, and the apoptotic cells means the dead cells. The ad-CALR promote cell growth, and miR-206 induce cellular apoptosis. The detail of this description was showed on page 7, line 206.

6. Could the authors provide representative images of transwell assay?

Response: Dear Reviewer#1, thank you for your concern. We did not take image for the transwell assay, and only counted the metastatic cells under the microscope.

7. Many grammar mistakes and errors were noted. It is suggested that authors seek for professional editing service to improve the quality of language and the organization of the manuscript.

Response: Dear Reviewer#1, thanks for your comment! We have carefully re-written and checked the grammar mistakes and errors in the full-text. We also asked an English native speaker for the correction.

Reviewer #2: On the whole, it is a valuable and meaningful study, and the design in this study is logical. However, some points should be explained as following before publication.

1. Its research significance and potential application value should be discussed in the discussion.

Response: Dear Reviewer#2, thank you for your suggestion! Some sentences on research significance and potential application value were added on the part of discussion. For example: “Our result not only clarified the mechanism of miR-206 regulating CALR expression in breast cancer stem cells, but also provided a novel target for the prevention and treatment of breast cancer. ” on page 10.

2. How to prove whether the transfection of MiR-206 mimics is successful?

Response: Dear Reviewer#2, thank you for your comment. Previously, the transfection of microRNA has been performed in our lab. The transfection rate is over 85%. We confirmed that our transfection method worked in this study.

3. What is the function of E-cadherin and N-cadherin in tumor progression? Why CALR exerted different effect on E-cadherin and N-cadherin?

Response: Dear Reviewer#2, as the description in the part of discussion on page 9 line 131, “EMT play an important role in the invasion and metastasis of malignant tumors, and main change in EMT in tumor cells is a decrease in the expression of the epithelial marker E-cadherin and an increase in the expression of the interstitial marker N-cadherin[19-21]. Overexpression of N-cadherin is closely related to the invasion and metastasis of a variety of epithelial-derived malignant tumors. When N-cadherin is over-expressed, the structure of tissue cells becomes loose, the adhesion between cells decreases, and then cells Invasion and transfer[22, 23].”

4. Why Ad-CALR can not reverse the effect of MiR-206 on E-cadherin and N-cadherin?

Response: Dear Reviewer#2, thank you for your concern! As we described in the part of result on page 8 line 219, “As seen in Fig.3B, overexpression of CALR enhanced the E-cadherin and reduced N-cadherin expression. miR-206 alone resulted in a decrease of E-cadherin and increase of N-cadherin. Co-transfection of Ad-CALR could partially reduce the biological effect of miR206 on EMT pathway. This result is consistent to our transwell assay”.

5. Did the Ad-EVI-1 treatment affect the effect of miR-206 mimics on cytotoxic effect, E-cadherin and N-cadherin?

Response: Dear Reviewer#2, in this study, we did investigate the effect of Ad-EVI-1 on miR-206 on cytotoxic effect and E-cadherin, N-cadherin expression. In fact, we only focused on the regulation of CALR expression in current study. In the future, we will investigate the function of EVI-1 in breast cancer stem cells.

6. Please explained the principle and mechanism of miR-206 mimics, Ad-CALR and Ad-EVI-1 transfected into cells, what is different with plasmid transfection?

Response: Dear Reviewer#2, thank you for your comment! Based on our result, the dose of miR-206 was chosen as 50nM. The amounted of Ad-CALR and Ad-EVI, we tested a series of doses, and the amount of 500ug of this plasmid could effectively overexpression the target protein. 1000 ug of plasmid is too toxic. Thus, 500 ug of plasmid was choose.

Reviewer #3: In this manuscript, Dapeng and co-authors describe the effect of miRNA-206 on the growth and migration of breast cancer cells and explore the underlying mechanism. Defining the role of miRNA-206 in breast cancer progression is useful to develop novel relevant prevention and/or therapy strategy . The authors did some work to validate their proposal. However, this study is not logically clear and some data is not convincing and need more detail work relating the underlying mechanism, particularly in relation to the relationship between miR-206, CALR and EVI-1 in their engaged signaling pathway.

Major comments

1. MM doesn’t clarify the origin of breast cancer stem cell, and more importantly these used stem cells should be fully/extensively characterized prior to functional assay, such as more stem cell markers besides CD44+/ CD24- should be employed to validate stem cells identity.

Response: Dear Reviewer#3, thank you for your suggestion! In our study, the breast cancer stem cells were stained and sorted by flow cytometry. We will investigate the function of breast cancer stem cell in the future study.

2. For the stem cells invasion/migration assay, it’s worth to include some representative images, that will be more visualized and convincing.

Response: Dear Reviewer#3, thank you for your comment! We did not take image for the transwell, and only count the cells under the microscope.

3. The final schematic is not consistent with the conclusion drawn in the text.

Response: Dear Reviewer#3, based on you comment! We have revised the schematic figure (as shown in Fig. 4F).

4. It is highly recommended to carefully go over the MM part to sort out some obvious incorrect description such as “transfected with 5ml Lipo 200”, repeated description, and incomplete method description.

Response: Dear Reviewer#3, thank you for your suggestion! We have carefully revised and edited the part of method.

5. Please include all the qPCR primers sequence used in this study.

Response: Dear Reviewer#3, according to your suggestion! The list of qPCR primers was listed in the part of method. As shown on page 6, line 193, “The primers as follow: CALR- F 5′-TGG TCC TGG TCC TGA TGT CG-3′ and CALR-R 5′-CTC TAC AGC TCG TCC TTG-3′; ACTN-F AGG TCA TCA CCA TCG GCA ACG A, ACTN-R GCT GTT GTA GGT GGT CTC GTG A.”

6. The EVI-1 background introduction should be incorporated.

Response: Dear Reviewer#3, based on your suggestion, the background of EVI-1 was inserted.

Minor comments

a. The cat # of Reagents/Cells/Instrument used in this study is missing.

Response: Dear Reviewer#3, thank you for your comment! The CatLog number of the agent was added in the part of method.

b. Please number all the rows of the text part, which will be much easier to be located during the reviewing process.

Response: Dear Reviewer#3, thank you for your suggestion! The row number was added in the revised manuscript.

c. The ms should be further polished regarding its writing in a professional way and tense using.

Response: Dear Reviewer#3, thanks for your comment! We have carefully re-written and checked the grammar mistakes and errors in the full-text. We also asked an English native speaker for the correction.

Attachment

Submitted filename: Point-to-Point Response letter to reviewer.docx

Click here for additional data file. (30.4KB, docx)

Decision Letter 1

Ning Wei

7 Sep 2022

MicroRNA-206 suppresses growth and metastasis of breast cancer stem cells via blocking EVI-1-mediated CALR expression

PONE-D-22-08405R1

Dear Dr. Zhang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Ning Wei

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The current version of this manuscript is acceptable.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this paper, the authors found that miR-206 mimics suppressed CALR expression, inhibited the proliferation and metastasis ability of breast cancer stem cells and finally induced cellular apoptosis. They further showed that this was correlated with suppression of EVI-1 signaling. The study was supported some solid data. And only a few issues need to be addressed.

1. Line 154-155, it should be actin instead of ACTN.

2. Please correct the uppercases and lowercases in the article.

3. For the Annexin V/PI staining, the authors can include Annexin V+ only part in Fig. E2.

Reviewer #2: (No Response)

Reviewer #3: Even the current version of the manuscript looks pretty decent, I would still strongly suggest performing extensive/full characterization of the stem cells in future study.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

**********

Acceptance letter

Ning Wei

12 Sep 2022

PONE-D-22-08405R1

MicroRNA-206 suppresses growth and metastasis of breast cancer stem cells via blocking EVI-1-mediated CALR expression

Dear Dr. Zhang:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Ning Wei

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 File. Raw images for western blots.

    (PDF)

    Click here for additional data file. (144.1KB, pdf)
    Attachment

    Submitted filename: Point-to-Point Response letter to reviewer.docx

    Click here for additional data file. (30.4KB, docx)

    Data Availability Statement

    All relevant data are within the paper and its Supporting Information files.

    Articles from PLoS ONE are provided here courtesy of PLOS

    RESOURCES