Macrophage-secreted exosomes inhibit breast cancer cell migration via the miR-101-3p/DLG5 axis

Abstract

Objective: To investigate the role of macrophages in regulating breast cancer cell migration and its related mechanisms. Methods: Human leukemia monocytic cell line THP-1-secreted exosomes were isolated using multi-step ultracentrifugation and verified using nanoparticle tracking analysis. Differentially expressed miRNAs were identified using RNA sequencing. Overexpression of inhibitors of hsa-miR-101-3p in breast cancer MDA-MB-231 cells was performed by infecting their lentiviral constructs. The luciferase reporter assay was used to evaluate the interaction of DLG5 and miR-101. DGL5 expression was detected using qRT-PCR and Western blot analyses. Results: The migration of breast cancer cells was significantly inhibited after addition of exosomes. RNA sequencing results showed that miR-101-3p expression was significantly upregulated. Targetscan analysis predicted that miR-101-3p could target DLG5, and this prediction was verified using the luciferase assay. The addition of the miR-101-3p precursor significantly increased the expression of miR-101-3p, and the mRNA and protein levels of DLG5 were suppressed. In contrast, inhibiting the expression of miR-101-3p increased the mRNA and protein levels of DLG5. Furthermore, the scratch assay showed that inhibiting miR-101-3p could promote the migration of MDA-MB-231 cells. Conclusions: Macrophage exosomes can inhibit the migration of breast cancer cells, and increasing the expression of miR-101-3p to inhibit DLG5 expression may play an important role in this process, which needs further investigation.

Keywords

Micro-RNA / Tumor-associated macrophages / Exosomes / Breast cancer / DLG5

Conflict of interest statement

The authors declare that they have no competing interests.

Funding

This project was supported by the Key Research and Development Program of Hainan Province (ZDYF2020139, ZDYF2018158) and the Science and Technology Funding Project of Hainan Province (821MS129).

Data availability statement

The data supporting the findings of this study are available from the corresponding authors upon request.

Authors’ contributions

YL conceptualized the study. CW, JX, YZ, SY, and QH performed experiments. YL and RY designed the experiments. YL, CW, and JX supervised experiments. All authors analyzed data and contributed to discussion. CW and YL wrote the paper. JX was the guarantor of this work and had full access to all the data in the study and took responsibility for the integrity of the data and the accuracy of the data analysis. All authors read and approved the final manuscript.

References

[1] Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2021; 71(3): 209-249.

[2] Hanker AB, Sudhan DR, Arteaga CL. Overcoming endocrine resistance in breast cancer. Cancer Cell 2020; 37(4): 496-513.

[3] Xu S, He X, Zhang L, Wang G, Luo T, Xu X, et al. Negative regulatory effect of microRNA-100 on the proliferation of breast cells and breast cancer cells. Acta Universitatis Medicinalis Anhui 2020; 55(2): 177-182.

[4] Sangpairoj K, Settacomkul R, Siangcham T, Meemon K, Niamnont N, Sornkaew N, et al. Hexadecanoic acid-enriched extract of Halymenia durvillei induces apoptotic and autophagic death of human triple-negative breast cancer cells by upregulating ER stress. Asian Pac J Trop Biomed 2022; 12(3): 132-140.

[5] Buranrat B, Junking M. Piperine suppresses growth and migration of human breast cancer cells through attenuation of Rac1 expression. Asian Pac J Trop Biomed 2022; 12(1): 39-46.

[6] Cansaran-Duman D, Tanman Ü, Yangın S, Atakol O. The comparison of miRNAs that respond to anti-breast cancer drugs and usnic acid for the treatment of breast cancer. Cytotechnology 2020; 72(6): 855-872.

[7] Han L, Chen WJ, Xia YM, Song YR, Zhao Z, Cheng H, et al. MiR-101 inhibits the proliferation and metastasis of lung cancer by targeting zinc finger E-box binding homeobox 1. Am J Transl Res 2018; 10(4): 1172-1183.

[8] Zhao H, Cao H, Ning F. Mechanism of microRNA-101 targeting pre-B-cell leukemia transcription factor 3 affecting the proliferation and apoptosis of acute T lymphocyte leukzemia cells. Chin J Cancer Prev Treat 2020; 27(20): 1605-1615.

[9] Franklin RA, Li MO. Ontogeny of tumor-associated macrophages and its implication in cancer regulation. Trends Cancer 2016; 2(1): 20-34.

[10] Cheng H, Wang Z, Fu L, Xu T. Macrophage polarization in the development and progression of ovarian cancers: An overview. Front Oncol 2019; 9: 421.

[11] Pegtel DM, Gould SJ. Exosomes. Annu Rev Biochem 2019; 88: 487-514.

[12] Zhang J, Li S, Li L, Li M, Guo C, Yao J, et al. Exosome and exosomal microRNA: Trafficking, sorting, and function. Genomics Proteomics Bioinformatics 2015; 13(1): 17-24.

[13] Yang F, Ning Z, Ma L, Liu W, Shao C, Shu Y, et al. Exosomal miRNAs and miRNA dysregulation in cancer-associated fibroblasts. Mol Cancer 2017; 16(1): 148.

[14] Liu J, Li J, Li P, Wang Y, Liang Z, Jiang Y, et al. Loss of DLG5 promotes breast cancer malignancy by inhibiting the Hippo signaling pathway. Sci Rep 2017; 7: 42125. doi: 10.1038/srep42125.

[15] Guen VJ, Chavarria TE, Kröger C, Ye X, Weinberg RA, Lees JA. EMT programs promote basal mammary stem cell and tumor-initiating cell stemness by inducing primary ciliogenesis and Hedgehog signaling. Proc Natl Acad Sci U S A 2017; 114(49): e10532-e10539.

[16] Wen S, Tan W, Liu W. Research progress of microRNA-34a in malignant tumors. Chin J Clin Res (Chin) 2020; 33(10): 1432-1434, 1438.

[17] Park J, Cho M, Cho J, Kim EE, Song EJ. MicroRNA-101-3p suppresses cancer cell growth by inhibiting the USP47-induced deubiquitination of RPL11. Cancers (Basel) 2022; 14(4). doi: 10.3390/cancers14040964.

[18] Wang H, Xiao R, Yang B. MiR-101-3p suppresses progression of cervical squamous cell carcinoma by targeting and down-regulating KPNA2. Technol Cancer Res Treat 2021; 20. doi: 10.1177/15330338211055948.

[19] Gu Z, You Z, Yang Y, Ding R, Wang M, Pu J, et al. Inhibition of MicroRNA miR-101-3p on prostate cancer progression by regulating Cullin 4B (CUL4B) and PI3K/AKT/mTOR signaling pathways. Bioengineered 2021; 12(1): 4719-4735.

[20] Wang S, Sun H, Zhang S, Chen L, Chen X, Li W. lncRNA TDRG1 promotes proliferation, invasion and migration of colorectal cancer cells by regulating miR-101-3p. Chin J Pathophysiol 2021; 37(5): 879-884.

[21] Guo X, Chen M, Cao L, Hu Y, Li X, Zhang Q, et al. Cancer-associated fibroblasts promote migration and invasion of non-small cell lung cancer cells via miR-101-3p mediated VEGFA secretion and AKT/eNOS pathway. Front Cell Dev Biol 2021; 9. doi: 10.3389/fcell.2021.764151.

[22] Bao RF, Shu YJ, Hu YP, Wang XA, Zhang F, Liang HB, et al. miR-101 targeting ZFX suppresses tumor proliferation and metastasis by regulating the MAPK/Erk and Smad pathways in gallbladder carcinoma. Oncotarget 2016; 7(16): 22339-22354.

[23] Smolen GA, Zhang J, Zubrowski MJ, Edelman EJ, Luo B, Yu M, et al. A genome-wide RNAi screen identifies multiple RSK-dependent regulators of cell migration. Genes Dev 2010; 24(23): 2654-2665.