The role of miR-133a in silibinin-mediated inhibition of the PI3K/AKT/mTOR pathway in MCF-7 breast carcinoma cells

Document Type : Original article

Authors

1 Department of Clinical Biochemistry, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran

2 Molecular Medicine Research Center, Hamadan University of Medical Science, Hamadan, Iran

Abstract

Breast cancer is particularly severe in women. Research highlights the crucial role of miRNAs in key cellular processes, showcasing their intricate interactions with the oncogenic PI3K/AKT/mTOR (PAM) signaling pathway and underscoring their significant role as tumor suppressors. The effect of silibinin on cell growth and survival was evaluated using an MTT assay. Bioinformatics analysis identified putative miR-133a targets inside the PAM pathway. After incubating MCF-7 cells with silibinin, we measured miR-133a, EGFR, PI3K, AKT, PTEN, and mTOR expression levels using qRT-PCR. Furthermore, protein expression levels of mTOR were assessed using Western blotting. The MTT experiment displayed that silibinin effectively inhibits MCF-7 cell proliferation in a time- and dose-dependent manner. Silibinin's IC50 value, determined at 370 μM after 48 hours, was established. qRT-PCR analysis at this IC50 concentration highlighted reduced expression of EGFR, PI3K, AKT, PTEN, and mTOR mRNAs, alongside increased miR-133a expression. Notably, miR-133a exhibited a negative correlation with both EGFR and PIK3C2A expression. Furthermore, western blotting confirmed silibinin's capacity to diminish p-mTOR protein levels, the ultimate element of the PAM signaling pathway. The findings enhance comprehension of silibinin's impact on PAM signaling and miR-133a expression, offering promise for targeted therapies in disrupting oncogenic pathways in MCF-7 breast cancer cells. This insight could advance breast cancer treatment strategies.

Keywords

References

  1. WHO. WHO launches new roadmap on breast cancer. The Global Breast Cancer Initiative (GBCI). 2023. p.1-3.
  2. WHO. Global breast cancer initiative implementation framework: Assessing, strengthening and scaling up of services for the early detection and management of breast cancer. World Health Organization. 2023;118 p.
  3. Ortega MA, Fraile-Martínez O, Asúnsolo Á, Buján J, García-Honduvilla N, Coca S. Signal transduction pathways in breast cancer: The important role of PI3K/Akt/mTOR. J Oncol 2020; 2020:9258396.
  4. Zhang X, Xing C, Guan W, Chen L, Guo K, Yu A, Xie K. Clinicopathological and prognostic significance of nestin expression in patients with breast cancer: A systematic review and meta-analysis. Cancer Cell Int 2020;20:169.
  5. Hua YT, Xu WX, Li H, Xia M. Emerging roles of MiR-133a in human cancers. J Cancer 2021;12:198-206.
  6. Flores-Pérez A, Marchat LA, Rodríguez-Cuevas S, Bautista VP, Fuentes-Mera L, Romero-Zamora D, Maciel-Dominguez A, de la Cruz OH, Fonseca-Sánchez M, Ruíz-García E, la Vega HA, López-Camarillo C. Suppression of cell migration is promoted by miR-944 through targeting of SIAH1 and PTP4A1 in breast cancer cells. BMC Cancer 2016;16:379.
  7. Noh EM, Yi MS, Youn HJ, Lee BK, Lee YR, Han JH, Yu HN, Kim JS, Jung SH. Silibinin enhances ultraviolet B-induced apoptosis in MCF-7 human breast cancer cells. J Breast Cancer 2011;14:8-13.
  8. Zheng N, Zhang P, Huang H, Liu W, Hayashi T, Zang L, Zhang Y, Liu L, Xia M, Tashiro SI, Onodera S, Ikejima T. ERα down-regulation plays a key role in silibinin-induced autophagy and apoptosis in human breast cancer MCF-7 cells. J Pharmacol Sci 2015;128: 97-107.
  9. Binienda A, Ziolkowska S, Pluciennik E. The anticancer properties of silibinin: Its molecular mechanism and therapeutic effect in breast cancer. Anti Cancer Agents Med Chem 2019;20:1787-1796.
  10. Tuli HS, Garg VK, Bhushan S, Uttam V, Sharma U, Jain A, Sak K, Yadav V, Lorenzo JM, Dhama K, Behl T, Sethi G. Natural flavonoids exhibit potent anticancer activity by targeting microRNAs in cancer: A signature step hinting towards clinical perfection. Transl Oncol 2023;27:101596.
  11. Jahanafrooz Z, Motamed N, Rinner B, Mokhtarzadeh A, Baradaran B. Silibinin to improve cancer therapeutic, as an apoptotic inducer, autophagy modulator, cell cycle inhibitor, and microRNAs regulator. Life Sci 2018;213:236-247.
  12. Du YM, Wang YB. MiR-637 inhibits proliferation and invasion of hepatoma cells by targeted degradation of AKT1. Eur Rev Med Pharmacol Sci 2019;23:567-575.
  13. He H, Tian W, Chen H, Jiang K. MiR-944 functions as a novel oncogene and regulates the chemoresistance in breast cancer. Tumour Biol 2016;37:1599-1607.
  14. Rahmani F, Ferns GA, Talebian S, Nourbakhsh M, Avan A, Shahidsales S. Role of regulatory miRNAs of the PI3K/AKT signaling pathway in the pathogenesis of breast cancer. Gene 2020;737:144459.
  15. Łukasiewicz S, Czeczelewski M, Forma A, Baj J, Sitarz R, Stanisławek A. Breast cancer—epidemiology, risk factors, classification, prognostic markers, and current treatment strategies—An updated review. Cancers (Basel) 2021;13:4287.
  16. Boojar MMA, Boojar MMA, Golmohammad S. Overview of Silibinin anti-tumor effects. J Herb Med. 2020;23:100375.
  17. Fruman DA, Chiu H, Hopkins BD, Bagrodia S, Cantley LC, Abraham RT. The PI3K pathway in human disease. Cell 2017;170:605-635.
  18. Miricescu D, Totan A, Stanescu-Spinu II, Badoiu SC, Stefani C, Greabu M. PI3K/AKT/ mTOR signaling pathway in breast cancer: From molecular landscape to clinical aspects. Int J Mol Sci 2020;22:173.
  19. Tewari D, Patni P, Bishayee A, Sah AN, Bishayee A. Natural products targeting the PI3K-Akt-mTOR signaling pathway in cancer: A novel therapeutic strategy. Semin Cancer Biol 2022;80:1-17.
  20. Xing Y, Lin NU, Maurer MA, Chen H, Mahvash A, Sahin A, Akcakanat A, Li Y, Abramson V, Litton J, Chavez-Macgregor M, Valero V, Piha-Paul SA, Hong D, Do KA, Tarco E, Riall D, Eterovic AK, Wulf GM, Cantley LC, Mills GB, Doyle LA, Winer E, Hortobagyi GN, Gonzalez-Angulo AM, Meric-Bernstam F. Phase II trial of AKT inhibitor MK-2206 in patients with advanced breast cancer who have tumors with PIK3CA or AKT mutations, and/or PTEN loss/PTEN mutation. Breast Cancer Res 2019;21:78.
  21. Kim S, Han J, Kim JS, Kim JH, Choe JH, Yang JH, Nam SJ, Lee JE. Silibinin suppresses EGFR ligand-induced CD44 expression through inhibition of EGFR activity in breast cancer cells. Anticancer Res 2011;31:3767-3773.
  22. Liang L, Li L, Zeng J, Gao Y, Chen YL, Wang ZQ, Wang XY, Chang LS, He D. Inhibitory effect of silibinin on EGFR signal-induced renal cell carcinoma progression via suppression of the EGFR/MMP-9 signaling pathway. Oncol Rep 2012;28:999-1005.
  23. Li Y, Zhang C, Cai D, Chen C, Mu D. Silibinin inhibits migration and invasion of the rhabdoid tumor G401 cell line via inactivation of the PI3K/Akt signaling pathway. Oncol Lett 2017;14:8035-8041.
  24. Jahanafrooz Z, Motameh N, Bakhshandeh B. Comparative evaluation of silibinin effects on cell cycling and apoptosis in human breast cancer MCF-7 and T47D cell lines. Asian Pac J Cancer Prev 2016;17:2661-2665.
  25. Jahanafrooz Z, Motamed N, Bakhshandeh B. Effects of miR-21 downregulation and silibinin treatment in breast cancer cell lines. Cytotechnology 2017;69:667-680.
  26. Noh EM, Yi MS, Youn HJ, Lee BK, Lee YR, Han JH, Yu HN, Kim JS, Jung SH. Silibinin enhances ultraviolet B-induced apoptosis in mcf-7 human breast cancer cells. J Breast Cancer 2011;14:8-13.
  27. Chen Y, Liu Z, Wang H, Tang Z, Liu Y, Liang Z, Deng X, Zhao M, Fu Q, Li L, Cai H, Xie W, Fang W. VPS33B negatively modulated by nicotine functions as a tumor suppressor in colorectal cancer. Int J cancer 2020;146:496-509.
  28. Cui W, Zhang S, Shan C, Zhou L, Zhou Z. microRNA‐133a regulates the cell cycle and proliferation of breast cancer cells by targeting epidermal growth factor receptor through the EGFR/A kt signaling pathway. FEBS J 2013;280:3962-3974.
  29. Guo N, Zhao Y, Zhang W, Li S, Li S, Yu J. MicroRNA‑133a downregulated EGFR expression in human non‑small cell lung cancer cells via AKT/ERK signaling. Oncol Lett 2018;16:6045-6050.
  30. Tang Y, Pan J, Huang S, Peng X, Zou X, Luo Y, Ren D, Zhang X, Li R, He P, Wa Q. Downregulation of miR-133a-3p promotes prostate cancer bone metastasis via activating PI3K/AKT signaling. J Exp Clin Cancer Res 2018;37:160.
  31. Maleki Zadeh M, Motamed N, Ranji N, Majidi M, Falahi F. Silibinin-induced apoptosis and downregulation of microRNA-21 and microRNA-155 in MCF-7 human breast cancer cells. J Breast Cancer 2016;19:45-52.
  32. Yazdi Rouholamini SE, Moghassemi S, Maharat Z, Hakamivala A, Kashanian S, Omidfar K. Effect of silibinin-loaded nano-niosomal coated with trimethyl chitosan on miRNAs expression in 2D and 3D models of T47D breast cancer cell line. Artif Cells Nanomed Biotechnol 2018;46:524-535.
  33. Chakrabarti M, Ray SK. Anti-tumor activities of luteolin and silibinin in glioblastoma cells: overexpression of miR-7-1-3p augmented luteolin and silibinin to inhibit autophagy and induce apoptosis in glioblastoma in vivo. Apoptosis 2016;21:312-328.
  34. Son SW, Lee HY, Moeng S, Kuh HJ, Choi SY, Park JK. Participation of microRNAs in the treatment of cancer with phytochemicals. Molecules 2020;25:4701.

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