Inducing Apoptosis and Decreases Cell Proliferation in Human Breast Cancer Cells through miR-182-5p Blockage Caused by Locked Nucleic Acid

Document Type : Original Article (s)

Authors

1 Assistant Professor, Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

2 PhD Student, Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

Abstract

Background: MicroRNAs (miRNAs) are single strand and short non-coding RNAs involved in post-transcription expression of genes. MiRNAs exhibited a substantial role in numerous cellular processes including cell proliferation, differentiation, cell cycle, apoptosis, and cancer development by negative regulation of tumor suppressor or oncogenic genes. Breast cancer is one of the most common cancers in world. Several studies reveal that miR-182-5p is up-regulated in breast cancer.Methods: MiR-182-5p blockage was performed using locked nucleic acid (LNA) technology in human breast cancer cell line (MCF-7). After blockage, miR-182-5p expression, cell proliferation, apoptosis and necrosis were measured. MiR-182-5p expression was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) at 24, 48, and 72 hour after locked nucleic acid anti-miR-182-5p transfection. Moreover, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and annexin/propidium iodide staining were evaluated.Findings: MiR-182-5p expression decreased at 24, 48, and 72 hours after transfection in the locked nucleic acid group compared to control groups. Cell viability was statistically different between locked nucleic acid and control groups. In the locked nucleic acid group, due to miR-182-5p inhibition, apoptosis ratio increased more than the other groups. Similarly, necrosis ratio in the locked nucleic acid group increased more than the other groups.Conclusion: In this study, miR-182-5p blockage reduced cell viability in MCF-7 cells chiefly due to induction of apoptosis and necrosis. Our results can help translational medicine for investigation on antisense therapy in human breast cancer.

Keywords

References

  1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin 2016; 66(1): 7-30.
  2. Dvinge H, Git A, Graf S, Salmon-Divon M, Curtis C, Sottoriva A, et al. The shaping and functional consequences of the microRNA landscape in breast cancer. Nature 2013; 497(7449): 378-82.
  3. Curtis C, Shah SP, Chin SF, Turashvili G, Rueda OM, Dunning MJ, et al. The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature 2012; 486(7403): 346-52.
  4. Le QJ, Caldas C. Micro-RNAs and breast cancer. Mol Oncol 2010; 4(3): 230-41.
  5. Buffa FM, Camps C, Winchester L, Snell CE, Gee HE, Sheldon H, et al. microRNA-associated progression pathways and potential therapeutic targets identified by integrated mRNA and microRNA expression profiling in breast cancer. Cancer Res 2011; 71(17): 5635-45.
  6. Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours. Nature 2012; 490(7418): 61-70.
  7. Lyng MB, Laenkholm AV, Sokilde R, Gravgaard KH, Litman T, Ditzel HJ. Global microRNA expression profiling of high-risk ER+ breast cancers from patients receiving adjuvant tamoxifen mono-therapy: a DBCG study. PLoS One 2012; 7(5): e36170.
  8. Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo R, Sabbioni S, et al. MicroRNA gene expression deregulation in human breast cancer. Cancer Res 2005; 65(16): 7065-70.
  9. Iorio MV, Croce CM. MicroRNAs in cancer: small molecules with a huge impact. J Clin Oncol 2009; 27(34): 5848-56.
  10. Yan LX, Huang XF, Shao Q, Huang MY, Deng L, Wu QL, et al. MicroRNA miR-21 overexpression in human breast cancer is associated with advanced clinical stage, lymph node metastasis and patient poor prognosis. RNA 2008; 14(11): 2348-60.
  11. Qian B, Katsaros D, Lu L, Preti M, Durando A, Arisio R, et al. High miR-21 expression in breast cancer associated with poor disease-free survival in early stage disease and high TGF-beta1. Breast Cancer Res Treat 2009; 117(1): 131-40.
  12. Scott GK, Goga A, Bhaumik D, Berger CE, Sullivan CS, Benz CC. Coordinate suppression of ERBB2 and ERBB3 by enforced expression of micro-RNA miR-125a or miR-125b. J Biol Chem 2007; 282(2): 1479-86.
  13. Iorio MV, Casalini P, Piovan C, Di LG, Merlo A, Triulzi T, et al. microRNA-205 regulates HER3 in human breast cancer. Cancer Res 2009; 69(6): 2195-200.
  14. Krishnan K, Steptoe AL, Martin HC, Wani S, Nones K, Waddell N, et al. MicroRNA-182-5p targets a network of genes involved in DNA repair. RNA 2013; 19(2): 230-42.
  15. Cloonan N, Brown MK, Steptoe AL, Wani S, Chan WL, Forrest AR, et al. The miR-17-5p microRNA is a key regulator of the G1/S phase cell cycle transition. Genome Biol 2008; 9(8): R127.
  16. Cho WC, Chow AS, Au JS. Restoration of tumour suppressor hsa-miR-145 inhibits cancer cell growth in lung adenocarcinoma patients with epidermal growth factor receptor mutation. Eur J Cancer 2009; 45(12): 2197-206.
  17. Myatt SS, Wang J, Monteiro LJ, Christian M, Ho KK, Fusi L, et al. Definition of microRNAs that repress expression of the tumor suppressor gene FOXO1 in endometrial cancer. Cancer Res 2010; 70(1): 367-77.
  18. Hirata H, Ueno K, Shahryari V, Deng G, Tanaka Y, Tabatabai ZL, et al. MicroRNA-182-5p promotes cell invasion and proliferation by down regulating FOXF2, RECK and MTSS1 genes in human prostate cancer. PLoS One 2013; 8(1): e55502.
  19. Liu Z, Liu J, Segura MF, Shao C, Lee P, Gong Y, et al. MiR-182 overexpression in tumourigenesis of high-grade serous ovarian carcinoma. J Pathol 2012; 228(2): 204-15.
  20. Lei R, Tang J, Zhuang X, Deng R, Li G, Yu J, et al. Suppression of MIM by microRNA-182 activates RhoA and promotes breast cancer metastasis. Oncogene 2014; 33(10): 1287-96.
  21. Liu H, Wang Y, Li X, Zhang YJ, Li J, Zheng YQ, et al. Expression and regulatory function of miRNA-182 in triple-negative breast cancer cells through its targeting of profilin 1. Tumour Biol 2013; 34(3): 1713-22.
  22. Guttilla IK, White BA. Coordinate regulation of FOXO1 by miR-27a, miR-96, and miR-182 in breast cancer cells. J Biol Chem 2009; 284(35): 23204-16.
  23. Parr C, Jiang WG. Metastasis suppressor 1 (MTSS1) demonstrates prognostic value and anti-metastatic properties in breast cancer. Eur J Cancer 2009; 45(9): 1673-83.
  24. Tarasov V, Jung P, Verdoodt B, Lodygin D, Epanchintsev A, Menssen A, et al. Differential regulation of microRNAs by p53 revealed by massively parallel sequencing: miR-34a is a p53 target that induces apoptosis and G1-arrest. Cell Cycle 2007; 6(13): 1586-93.
  25. Suzuki HI, Yamagata K, Sugimoto K, Iwamoto T, Kato S, Miyazono K. Modulation of microRNA processing by p53. Nature 2009; 460(7254): 529-33.
  26. Segura MF, Hanniford D, Menendez S, Reavie L, Zou X, Alvarez-Diaz S, et al. Aberrant miR-182 expression promotes melanoma metastasis by repressing FOXO3 and microphthalmia-associated transcription factor. Proc Natl Acad Sci USA 2009; 106(6): 1814-9.
  27. Li AY, Boo LM, Wang SY, Lin HH, Wang CC, Yen Y, et al. Suppression of nonhomologous end joining repair by overexpression of HMGA2. Cancer Res 2009; 69(14): 5699-706.
  28. Stoffel M, Poy MN, Tuschl TH. MicroRNA and methods for inhibiting same. Publication No. US7365058 B2 [Patents]. 2008.
  29. Iorio MV, Croce CM. MicroRNA dysregulation in cancer: diagnostics, monitoring and therapeutics. A comprehensive review. EMBO Mol Med 2012; 4(3): 143-59.
  30. Orom UA, Kauppinen S, Lund AH. LNA-modified oligonucleotides mediate specific inhibition of microRNA function. Gene 2006; 372: 137-41.
Journal of Isfahan Medical School
Volume 35, Issue 418 - Serial Number 418
January and February 2017
Pages 57-63

Files

History

  • Receive Date: 05 February 2017
  • Revise Date: 30 April 2024
  • Accept Date: 06 April 2022

Share

How to cite

Statistics