EFFECT of CURCUMIN on BREAST CANCER CELLS THROUGH miR-145-5p AND ITS TARGET GENES

Öz

Objective: Curcumin is considered an epigenetic regulator with anticancer effects. The micro-RNA (miRNA) miR-145-5p is a tumor suppressor that shows low expression levels in various cancers, including breast cancer (BC). This study aims to investigate whether curcumin inhibits MCF-7 human BC cell line proliferation and migration by regulating miR-145-5p and its possible target genes. Material and Method: MCF-7 cells were treated with curcumin and its solvent control. Additionally, cells were transfected with an miR-145-5p mimic and a non-targeting miRNA mimic. Cell viability was then evaluated, and the scratch wound assay was used to assess cell migration. The study predicts the miR-145- 5p putative target genes by searching for overlapping genes in the miRNet and miRTarBase v8 databases via the overexpressed genes in the BC tissue samples in the Cancer Genome Atlas (TCGA) datasets. Expression levels of miR-145-5p and the selected genes were detected using the quantitative real-time polymerase chain reaction (qRT-PCR). The 2-ΔΔCt method was used for the quantification analysis, with p<0.05 being considered statistically significant. Result: Curcumin treatment and overexpression of miR-145- 5p via the transfection of an miR-145-5p mimic significantly decreased the proliferation and migration of MCF-7 cells. Moreover, curcumin treatment significantly increased the expression of miR-145-5p. The possible target genes of miR-145- 5p (i.e., MCM2, MMP1, MMP9, EEF1A2) were downregulated in curcumin-treated MCF-7 cells. Additionally, miR-145-5p mimictransfected cells showed low expression levels of the MCM2, MMP1, and MMP9 genes. Conclusion: Curcumin inhibits the proliferation and migration of MCF-7 cells by acting on miR-145-5p and its possible target genes.

Anahtar Kelimeler

• Breast cancer
• MCF-7 cell line
• curcumin
• miR-145- 5p
• target genes

KURKUMİNİN miR-145-5p VE HEDEF GENLERİ ÜZERİNDEN MEME KANSERİ HÜCRELERİNE ETKİSİ

Öz

Amaç: Kurkumin anti-kanser etkileri olan bir epigenetik regülatör olarak kabul edilmektedir. miR-145-5p, meme kanseri (MK) dahil olmak üzere bir çok kanserde ekspresyon düzeyi düşük olan bir tümör baskılayıcı mikroRNA'dır (miRNA). Bu çalışmada, kurkuminin miR-145-5p’yi ve olası hedef genlerini regüle ederek MCF-7 insan MK hücre hattının proliferasyonunu ve göçünü inhibe edip etmediğinin araştırılması amaçlanmıştır. Gereç ve Yöntem: MCF-7 hücreleri kurkumin ve onun solvent kontrolü ile muamele edildi. Ayrıca hücreler miR-145-5p mimiği ve negatif kontrol miRNA mimiği ile transfekte edildi. Hücre canlılığı değerlendirildi ve hücre göçü scratch yara (wound) testi kullanılarak değerlendirildi. miR-145-5p’nin potansiyel hedefleri, MK doku örneklerinde aşırı eksprese edilen genlerle miRNet ve miRTarBase v8 veri-tabanları ile örtüşen genlerin Kanser Genom Atlası (TCGA) datasetlerinde araştırılması yoluyla belirlendi. miR-145-5p ve seçilen genlerin ekspresyon düzeyleri, kantitatif gerçek-zamanlı polimeraz zincir reaksiyonu (qRT-PCR) ile tespit edildi. Kantifikasyon analizi için 2-ΔΔCt yöntemi kullanıldı. p-değeri <0,05 istatistiksel olarak anlamlı kabul edildi. Bulgular: Hem kurkumin ile muamele edilmiş hem de miR-145-5p mimiği ile transfekte edilmiş MCF-7 hücrelerinde, proliferasyonun ve göçün, kontrollere kıyasla azaldığı gözlenmiştir. Ayrıca, kurkumin ile muamele edilen hücrelerde miR-145-5p'nin ekspresyonunun anlamlı düzeyde arttığı görülmüştür. miR-145-5p’nin olası hedef genlerinin, MCM2, MMP1, MMP9 ve EEF1A2, kurkumin ile muamele edilmiş MCF-7 hücrelerinde ekspresyon düzeyinin azaldığı saptanmıştır. Bunun yanı sıra, miR-145-5p mimiği ile transfekte edilmiş hücrelerde, MCM2, MMP1 ve MMP9 genlerinin ekspresyon düzeyinin düşük olduğu belirlenmiştir. Sonuç: Kurkuminin, miR-145-5p ve olası hedef genleri üzerinden etki ederek MCF-7 hücrelerinin hem proliferasyonunu hem de göçünü inhibe ettiği gösterilmiştir.

Anahtar Kelimeler

• Meme kanseri
• MCF-7 hücre hattı
• kurkumin
• miR-145-5p
• hedef genler

Kaynakça

1. 1. Wilkinson L, Gathani T. Understanding breast cancer as a global health concern. Br J Radiol 2022;95(1130):20211033. [CrossRef] google scholar
2. 2. Erdogan C, Suer I, Kaya M, Ozturk S, Aydin N, Kurt Z. Bioinformatics analysis of the potentially functional circRNA-miRNA-mRNA network in breast cancer. PLoS One 2024;19(4):e0301995. [CrossRef] google scholar
3. 3. Arnold M, Morgan E, Rumgay H, Mafra A, Singh D, Laversanne M, et al. Current and future burden of breast cancer: Global statistics for 2020 and 2040. Breast 2022;66:15-23. [CrossRef] google scholar
4. 4. Guofeng Q, Wei W, Wei D, Fan Z, Sinclair AJ, Chatwin CR. Bioimpedance analysis for the characterization of breast cancer cells in suspension. IEEE Trans Biomed Eng 2012;59(8):2321-9. [CrossRef] google scholar
5. 5. Liu Y, Sun H, Makabel B, Cui Q, Li J, Su C, et al. The targeting of non-coding RNAs by curcumin: Facts and hopes for cancer therapy (Review). Oncol Rep 2019;42(1):20-34. [CrossRef] google scholar
6. 6. Bagatir G, Kaya M, Suer I, Cefle K, Palanduz A, Palanduz S, et al. The effect of Anzer honey on X-ray induced genotoxicity in human lymphocytes: An in vitro study. Microsc Res Tech 2022;85(6):2241-50. [CrossRef] google scholar
7. 7. Zhang JY, Lin MT, Zhou MJ, Yi T, Tang YN, Tang SL, et al. Combinational Treatment of Curcumin and Quercetin against Gastric Cancer MGC-803 Cells in Vitro. Molecules 2015;20(6):11524-34. [CrossRef] google scholar
8. 8. Su J, Zhou X, Wang L, Yin X, Wang Z. Curcumin inhibits cell growth and invasion and induces apoptosis through down-regulation of Skp2 in pancreatic cancer cells. Am J Cancer Res 2016;6(9):1949-62. google scholar

9. 9. Kaya M, Abuaisha A, Suer I, Emiroglu S, Abanoz F, Palanduz S, et al. Turmeric Inhibits MDA-MB-231 Cancer Cell Proliferation, Altering miR-638-5p and Its Potential Targets. Eur J Breast Health 2024;20(2):102-9. [CrossRef] google scholar
10. 10. Kaya M, Suer I. The effect of miR-34a-5p on overexpressed AML associated genes. J Ist Faculty Med 2023;86(1):59-68. [CrossRef] google scholar
11. 11. Suer I, Kaya M. Is the AURKB Gene Involved in Aml Cell Proliferation Since It is Targeted by miR-34a-5p and let-7b-5p? Konuralp Medical Journal 2023;15(1):16-23. [CrossRef] google scholar
12. 12. Capik O, Sanli F, Kurt A, Ceylan O, Suer I, Kaya M, et al. CASC11 promotes aggressiveness of prostate cancer cells through miR-145/IGF1R axis. Prostate Cancer Prostatic Dis 2021;24(3):891-902. [CrossRef] google scholar
13. 13. Kaya M, Suer I, Ozgur E, Capik O, Karatas OF, Ozturk S, et al. miR-145-5p suppresses cell proliferation by targeting IGF1R and NRAS genes in multiple myeloma cells. Turkish Journal of Biochemistry. 2023;48(5):563-9. [CrossRef] google scholar
14. 14. Rajarajan D, Kaur B, Penta D, Natesh J, Meeran SM. miR-145-5p as a predictive biomarker for breast cancer stemness by computational clinical investigation. Comput Biol Med 2021;135:104601. [CrossRef] google scholar
15. 15. Tang W, Zhang X, Tan W, Gao J, Pan L, Ye X, et al. miR-145-5p Suppresses Breast Cancer Progression by Inhibiting SOX2. J Surg Res 2019;236:278-87. [CrossRef] google scholar
16. 16. Jiang Y, Wang D, Ren H, Shi Y, Gao Y. MiR-145-targeted HBXIP modulates human breast cancer cell proliferation. Thorac Cancer 2019;10(1):71-7. [CrossRef] google scholar
17. 17. Hajibabaei S, Sotoodehnejadnematalahi F, Nafissi N, Zeinali S, Azizi M. Aberrant promoter hypermethylation of miR-335 and miR-145 is involved in breast cancer PD-L1 overexpression. Sci Rep 2023;13(1):1003. [CrossRef] google scholar
18. 18. Zhu X, Zhu R. Curcumin suppresses the progression of laryngeal squamous cell carcinoma through the upregulation of miR-145 and inhibition of the PI3K/Akt/ mTOR pathway. Onco Targets Ther 2018;11:3521-31. [CrossRef] google scholar
19. 19. Shen Y, Han Z, Liu S, Jiao Y, Li Y, Yuan H. Curcumin Inhibits the Tumorigenesis of Breast Cancer by Blocking Tafazzin/Yes-Associated Protein Axis. Cancer Manag Res 2020;12:1493-502. [CrossRef] google scholar
20. 20. Chatterjee B, Ghosh K, Suresh L, Kanade SR. Curcumin ameliorates PRMT5-MEP50 arginine methyltransferase expression by decreasing the Sp1 and NF-YA transcription factors in the A549 and MCF-7 cells. Mol Cell Biochem 2019;455(1-2):73-90. [CrossRef] google scholar
21. 21. Liu JL, Pan YY, Chen O, Luan Y, Xue X, Zhao JJ, et al. Curcumin inhibits MCF-7 cells by modulating the NF-kB signaling pathway. Oncol Lett 2017;14(5):5581-4. [CrossRef] google scholar
22. 22. Tang Z, Yang Y, Chen W, Li E, Liang T. Demethylation at enhancer upregulates MCM2 and NUP37 expression predicting poor survival in hepatocellular carcinoma patients. J Transl Med 2022;20(1):49. [CrossRef] google scholar
23. 23. Ezure T, Sugahara M, Amano S. Senescent dermal fibroblasts negatively influence fibroblast extracellular matrix-related gene expression partly via secretion of complement factor D. Biofactors 2019;45(4):556-62. [CrossRef] google scholar
24. 24. Kang H, Jang SW, Pak JH, Shim S. Glaucine inhibits breast cancer cell migration and invasion by inhibiting MMP-9 gene expression through the suppression of NF-kB activation. Mol Cell Biochem 2015;403(1-2):85-94. [CrossRef] google scholar
25. 25. Worst TS, Waldbillig F, Abdelhadi A, Weis CA, Gottschalt M, Steidler A, et al. The EEF1A2 gene expression as risk predictor in localized prostate cancer. BMC Urol 2017;17(1):86. [CrossRef] google scholar
26. 26. Ifon ET, Pang AL, Johnson W, Cashman K, Zimmerman S, Muralidhar S, et al. U94 alters FN1 and ANGPTL4 gene expression and inhibits tumorigenesis of prostate cancer cell line PC3. Cancer Cell Int 2005;5:19. [CrossRef] google scholar
27. 27. Suer I, Karatas OF, Yuceturk B, Yilmaz M, Guven G, Buge O, et al. Characterization of stem-like cells directly isolated from freshly resected laryngeal squamous cell carcinoma specimens. Curr Stem Cell Res Ther 2014;9(4):347-53. [CrossRef] google scholar
28. 28. Tang Z, Kang B, Li C, Chen T, Zhang Z. GEPIA2: an enhanced web server for large-scale expression profiling and interactive analysis. Nucleic Acids Res 2019;47(W1):W556-w60. [CrossRef] google scholar
29. 29. Tomczak K, Czerwi n ska P, Wiznerowicz M. The Cancer Genome Atlas (TCGA): an immeasurable source of knowledge. Contemp Oncol (Pozn) 2015;19(1a):A68-77. [CrossRef] google scholar
30. 30. Chang L, Zhou G, Soufan O, Xia J. miRNet 2.0: network-based visual analytics for miRNA functional analysis and systems biology. Nucleic Acids Res 2020;48(W1):W244-51. [CrossRef] google scholar
31. 31. Huang HY, Lin YC, Cui S, Huang Y, Tang Y, Xu J, et al. miRTarBase update 2022: an informative resource for experimentally validated miRNA-target interactions. Nucleic Acids Res 2022;50(D1):D222-d30. [CrossRef] google scholar
32. 32. Martin FJ, Amode MR, Aneja A, Austine-Orimoloye O, Azov AG, Barnes I, et al. Ensembl 2023. Nucleic Acids Res 2023;51(D1):D933-d41. google scholar
33. 33. Stelzer G, Rosen N, Plaschkes I, Zimmerman S, Twik M, Fishilevich S, et al. The GeneCards Suite: From Gene Data google scholar Mining to Disease Genome Sequence Analyses. Curr Protoc Bioinformatics 2016;54:1.30.1-33. [CrossRef] google scholar
34. 34. Zhou Y, Zhou B, Pache L, Chang M, Khodabakhshi AH, Tanaseichuk O, et al. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nat Commun 2019;10(1):1523. [CrossRef] google scholar
35. 35. Pinero J, Ramırez-Anguita JM, Saüch-Pitarch J, Ronzano F, Centeno E, Sanz F, Furlong LI. The DisGeNET knowledge platform for disease genomics: 2019 update. Nucleic Acids Res 2020;48(D1):D845-55. [CrossRef] google scholar
36. 36. Lânczky A, Györffy B. Web-Based Survival Analysis Tool Tailored for Medical Research (KMplot): Development and Implementation. J Med Internet Res 2021;23(7):e27633. [CrossRef] google scholar
37. 37. Zogopoulos VL, Saxami G, Malatras A, Papadopoulos K, Tsotra I, Iconomidou VA, Michalopoulos I. Approaches in Gene Coexpression Analysis in Eukaryotes. Biology (Basel) 2022;11(7):1019. [CrossRef] google scholar
38. 38. Miller HE, Bishop AJR. Correlation AnalyzeR: functional predictions from gene co-expression correlations. BMC Bioinformatics 2021;22(1):206. [CrossRef] google scholar
39. 39. Sadoughi F, Maleki Dana P, Asemi Z, Yousefi B. Targeting microRNAs by curcumin: implication for cancer therapy. Crit Rev Food Sci Nutr 2022;62(28):7718-29. [CrossRef] google scholar
40. 40. Ding Y, Zhang C, Zhang J, Zhang N, Li T, Fang J, et al. miR-145 inhibits proliferation and migration of breast cancer cells by directly or indirectly regulating TGF-Ş1 expression. Int J Oncol 2017;50(5):1701-10. [CrossRef] google scholar
41. 41. Song X, Zhang M, Dai E, Luo Y. Molecular targets of curcumin in breast cancer (Review). Mol Med Rep 2019;19(1):23-9. [CrossRef] google scholar
42. 42. Liu T, Chi H, Chen J, Chen C, Huang Y, Xi H, et al. Curcumin suppresses proliferation and in vitro invasion of human prostate cancer stem cells by ceRNA effect of miR-145 and lncRNA-ROR. Gene 2017;631:29-38. [CrossRef] google scholar
43. 43. Samad A, Haque F, Nain Z, Alam R, Al Noman MA, Rahman Molla MH, et al. Computational assessment of MCM2 transcriptional expression and identification of the prognostic biomarker for human breast cancer. Heliyon 2020;6(10):e05087. [CrossRef] google scholar
44. 44. Zou J, Zhu L, Jiang X, Wang Y, Wang Y, Wang X, Chen B. Curcumin increases breast cancer cell sensitivity to cisplatin by decreasing FEN1 expression. Oncotarget 2018;9(13):11268-78. [CrossRef] google scholar
45. 45. Chabottaux V, Noel A. Breast cancer progression: insights into multifaceted matrix metalloproteinases. Clin Exp Metastasis 2007;24(8):647-56. [CrossRef] google scholar
46. 46. Akter T, Aziz MA, Islam MS, Sarwar MS. Association of MMP1 gene polymorphisms with breast cancer risk: A narrative review. Health Sci Rep 2023;6(10):e1607. [CrossRef] google scholar
47. 47. Qu J, Zhao X, Liu X, Sun Y, Wang J, Liu L, et al. Natriuretic peptide receptor a promotes breast cancer development by upregulating MMP9. Am J Cancer Res 2019;9(7):1415-28. google scholar
48. 48. Cheng L, Tan Z, Huang Z, Pan Y, Zhang W, Wang J. Expression Profile and Prognostic Values of Mini-Chromosome Maintenance Families (MCMs) in Breast Cancer. Med Sci Monit 2020;26:e923673. [CrossRef] google scholar
49. 49. Lim JP, Nair S, Shyamasundar S, Chua PJ, Muniasamy U, Matsumoto K, et al. Silencing Y-box binding protein-1 inhibits triple-negative breast cancer cell invasiveness via regulation of MMP1 and beta-catenin expression. Cancer Lett 2019;452:119-31. [CrossRef] google scholar
50. 50. Schveigert D, Cicenas S, Bruzas S, Samalavicius NE, Gudleviciene Z, Didziapetriene J. The value of MMP-9 for breast and non-small cell lung cancer patients’ survival. Adv Med Sci 2013;58(1):73-82. [CrossRef] google scholar