Enhanced Expression of miR-638-5p May Suppress Acute Myeloid Leukemia in vitro Cell Proliferation Through PGK1 and PIM1

Year 2025, Volume: 17 Issue: 1, 39 - 47, 27.03.2025

Simge Özkaynak , Ilknur Suer , Murat Kaya , Şükrü Öztürk

Abstract

Objective: miR-638-5p is a crucial tumor suppressor miRNA in several cancer types including, Acute Myeloid Leukemia (AML). This study aimed to analyze the role of miR-638-5p and its potential target genes in HL-60 and NB4 acute promyelocytic leukemia cell lines using in vitro method.
Method: After the miR-638-5p mimic transfection into AML cells, the effect on cell viability was examined by the WST-8 method, and the effect on apoptosis was measured via the Caspase-3 quantification method. In silico tools such as miRWalk, miRDB, and miRTarBase were used to select the possible target genes of miR-638-5p. The expression levels of selected genes were investigated by qRT-PCR. The overall survival (OS) rate of AML patients was explored via the BloodSpot database, the Enrichr tool was used for enrichment analysis, and correlation analysis was performed using the Correlation AnalyzeR tool.
Results: Decreased proliferation and increased apoptosis were determined in miR-638-5p mimic transfected cells compared to the controls. MECP2, PIM1, MEF2C, PGK1, and SPAG1 genes were selected as the potential targets of miR-638-5p for in vitro study. PGK1 and PIM1 expression levels were significantly suppressed in cells transfected with the miR-638-5p mimic. The OS investigation revealed that overexpression of MECP2, MEF2C, and PGK1 does not affect the survival of AML patients; however, overexpression of SPAG1 and PIM1 has a detrimental effect on AML survival. Also, a positive correlation was detected between PIM1 and PGK1 genes via enrichment analysis.
Conclusion: miR-638-5p may contribute to AML pathogenesis by targeting the PGK1 and PIM1 genes, and this situation may indicate its potential as a biomolecule for regulating cell proliferation in AML cells.

Keywords

miR-638-5p, Acute Myeloid Leukemia, HL-60, NB4, microRNA mimic transfection

Ethical Statement

Ethics committee approval is not required, patient samples were not incorporated, and only cell lines were included in this study.

Supporting Institution

This study was supported by the Research Fund of Istanbul University (Project No: 37151).

Project Number

Project No: 37151

miR-638-5p'nin Ekspresyon Artışı PGK1 ve PIM1 Aracılığıyla Akut Miyeloid Lösemide in vitro Hücre Proliferasyonunu Baskılayabilir

Abstract

Amaç: miR-638-5p, Akut Miyeloid Lösemi (AML) dahil olmak üzere çeşitli kanser türlerinde önemli bir tümör baskılayıcı miRNA'dır. Bu çalışma, miR-638-5p'nin ve potansiyel hedef genlerinin HL-60 ve NB4 akut promyelositik lösemi hücre hatları üzerindeki rolünü in vitro ortamda incelemeyi amaçlamaktadır.
Metod: miR-638-5p mimik transfeksiyonundan sonra AML hücrelerinin canlılığı üzerindeki etki WST-8 yöntemi ile apoptoz üzerindeki etki ise Caspase-3 kantifikasyon yöntemi ile incelendi. miRWalk, miRDB ve miRTarBase gibi in silico araçlar miR-638-5p'nin olası hedef genlerini seçmek için kullanıldı. Seçilen genlerin ifade düzeyleri qRT-PCR ile araştırıldı. AML hastalarının genel sağkalım (OS) oranı BloodSpot veritabanı üzerinden araştırıldı, zenginleştirme analizi için Enrichr aracı kullanıldı ve korelasyon analizi Correlation AnalyzeR aracı kullanılarak gerçekleştirildi.
Bulgular: Kontrollerle karşılaştırıldığında miR-638-5p mimik transfekte hücrelerde azalmış proliferasyon ve artmış apoptoz belirlendi. MECP2, PIM1, MEF2C, PGK1 ve SPAG1 genleri in vitro çalışma için miR-638-5p potansiyel hedefleri olarak seçildi. PGK1 ve PIM1 ekspresyon seviyeleri miR-638-5p mimic transfekte her iki hücrede de önemli ölçüde baskılandı. OS araştırması, MECP2, MEF2C ve PGK1'in aşırı ekspresyonunun AML hastalarının sağkalımını etkilemediğini; ancak SPAG1 ve PIM1'in aşırı ekspresyonunun AML sağkalımı üzerinde zararlı bir etkiye sahip olduğunu ortaya koydu. Ayrıca, zenginleştirme analizi yoluyla PIM1 ve PGK1 genleri arasında pozitif bir korelasyon tespit edildi.
Sonuç: miR-638-5p'nin PGK1 ve PIM1 genlerini hedef alarak AML patogenezine katkıda bulunabileceği ve bu durumun AML hücrelerinde hücre proliferasyonunu düzenleyen bir biyomolekül olarak potansiyeline işaret edebileceği düşünülmektedir.

Keywords

miR-638-5p, Akut Miyeloid Lösemi, HL-60, NB4, mikroRNA mimik transfeksiyonu

Project Number

Project No: 37151

References

• 1. De Kouchkovsky I, Abdul-Hay M. 'Acute myeloid leukemia: a comprehensive review and 2016 update'. Blood Cancer J. 2016;6(7):e441.
• 2. Lyengar V SA. Leukemia: StatPearls Publishing, Treasure Island (FL) 2020
• 3. Kaya M, Abuaisha A, Süer İ, Alptekin MS, Abanoz F, Emiroğlu S, et al. Overexpression of CDC25A, AURKB, and TOP2A Genes Could Be an Important Clue for Luminal A Breast Cancer. Eur J Breast Health. 2024;20(4):284-91.
• 4. Wang S, Xu J, Guo Y, Cai Y, Zhu W, Meng L, et al. Successful Transfection of MicroRNA Mimics or Inhibitors in a Regular Cell Line and in Primary Cells Derived from Patients with Rheumatoid Arthritis. Bio Protoc. 2023;13(18):e4823.
• 5. Kaya M, Abuaisha A, Suer I, Emiroglu S, Önder S, Onay Ucar E, et al. Let-7b-5p sensitizes breast cancer cells to doxorubicin through Aurora Kinase B. PLoS One. 2025;20(1):e0307420.
• 6. 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.
• 7. 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.
• 8. Wang J, Chen J, Sen S. MicroRNA as Biomarkers and Diagnostics. J Cell Physiol. 2016;231(1):25-30.
• 9. Condrat CE, Thompson DC, Barbu MG, Bugnar OL, Boboc A, Cretoiu D, et al. miRNAs as Biomarkers in Disease: Latest Findings Regarding Their Role in Diagnosis and Prognosis. Cells. 2020;9(2).
• 10. 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.
• 11. Kaya M. Bioinformatics evaluation of the circRNA–miRNA–mRNA axis in cervical squamous cell carcinoma. Exploration of Medicine. 2024;5:553-65.
• 12. Lee YS, Dutta A. MicroRNAs in cancer. Annu Rev Pathol. 2009;4:199-227.
• 13. Shi M, Jiang Y, Yang L, Yan S, Wang YG, Lu XJ. Decreased levels of serum exosomal miR-638 predict poor prognosis in hepatocellular carcinoma. J Cell Biochem. 2018;119(6):4711-6.
• 14. Zheng DH, Wang X, Lu LN, Chen DL, Chen JM, Lin FM, et al. MiR-638 serves as a tumor suppressor by targeting HOXA9 in glioma. Eur Rev Med Pharmacol Sci. 2018;22(22):7798-806.
• 15. Wang F, Lou J-f, Cao Y, Shi X-h, Wang P, Xu J, et al. miR-638 is a new biomarker for outcome prediction of non-small cell lung cancer patients receiving chemotherapy. Experimental & Molecular Medicine. 2015;47(5):e162-e.
• 16. Yao Y, Suo AL, Li ZF, Liu LY, Tian T, Ni L, et al. MicroRNA profiling of human gastric cancer. Mol Med Rep. 2009;2(6):963-70.
• 17. Bai Y, Chen C, Guo X, Ding T, Yang X, Yu J, et al. miR-638 in circulating leukaemia cells as a non-invasive biomarker in diagnosis, treatment response and MRD surveillance of acute promyelocytic leukaemia. Cancer Biomark. 2020;29(1):125-37.
• 18. Zhou J, Guo H, Yang Y, Zhang Y, Liu H. A meta-analysis on the prognosis of exosomal miRNAs in all solid tumor patients. Medicine (Baltimore). 2019;98(16):e15335.
• 19. Lin Y, Li D, Liang Q, Liu S, Zuo X, Li L, et al. miR-638 regulates differentiation and proliferation in leukemic cells by targeting cyclin-dependent kinase 2. J Biol Chem. 2015;290(3):1818-28.
• 20. Gíslason MH, Demircan GS, Prachar M, Furtwängler B, Schwaller J, Schoof EM, et al. BloodSpot 3.0: a database of gene and protein expression data in normal and malignant haematopoiesis. Nucleic Acids Res. 2024;52(D1):D1138-d42.
• 21. Kuleshov MV, Jones MR, Rouillard AD, Fernandez NF, Duan Q, Wang Z, et al. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res. 2016;44(W1):W90-7.
• 22. Miller HE, Bishop AJR. Correlation AnalyzeR: functional predictions from gene co-expression correlations. BMC Bioinformatics. 2021;22(1):206.
• 23. Almeida AM, Ramos F. Acute myeloid leukemia in the older adults. Leuk Res Rep. 2016;6:1-7.
• 24. Narayan N, Bracken CP, Ekert PG. MicroRNA-155 expression and function in AML: An evolving paradigm. Exp Hematol. 2018;62:1-6.
• 25. Weng H, Lal K, Yang FF, Chen J. The pathological role and prognostic impact of miR-181 in acute myeloid leukemia. Cancer Genet. 2015;208(5):225-9.
• 26. Garzon R, Volinia S, Liu CG, Fernandez-Cymering C, Palumbo T, Pichiorri F, et al. MicroRNA signatures associated with cytogenetics and prognosis in acute myeloid leukemia. Blood. 2008;111(6):3183-9.
• 27. Wang F, Lou JF, Cao Y, Shi XH, Wang P, Xu J, et al. miR-638 is a new biomarker for outcome prediction of non-small cell lung cancer patients receiving chemotherapy. Exp Mol Med. 2015;47:e162.
• 28. Lin QY, Wang JQ, Wu LL, Zheng WE, Chen PR. miR-638 represses the stem cell characteristics of breast cancer cells by targeting E2F2. Breast Cancer. 2020;27(1):147-58.
• 29. Bhattacharya A, Schmitz U, Raatz Y, Schonherr M, Kottek T, Schauer M, et al. miR-638 promotes melanoma metastasis and protects melanoma cells from apoptosis and autophagy. Oncotarget. 2015;6(5):2966-80.
• 30. Zhang J, Fei B, Wang Q, Song M, Yin Y, Zhang B, et al. MicroRNA-638 inhibits cell proliferation, invasion and regulates cell cycle by targeting tetraspanin 1 in human colorectal carcinoma. Oncotarget. 2014;5(23):12083-96.
• 31. Zhu DX, Zhu W, Fang C, Fan L, Zou ZJ, Wang YH, et al. miR-181a/b significantly enhances drug sensitivity in chronic lymphocytic leukemia cells via targeting multiple anti-apoptosis genes. Carcinogenesis. 2012;33(7):1294-301.
• 32. Chong ZX, Yeap SK, Ho WY. Dysregulation of miR-638 in the progression of cancers. Pathology - Research and Practice. 2021;220:153351.
• 33. Wang H, Yao H, Yi B, Kazama K, Liu Y, Deshpande D, et al. MicroRNA-638 inhibits human airway smooth muscle cell proliferation and migration through targeting cyclin D1 and NOR1. J Cell Physiol. 2018;234(1):369-81.
• 34. Zhao LY, Yao Y, Han J, Yang J, Wang XF, Tong DD, et al. miR-638 suppresses cell proliferation in gastric cancer by targeting Sp2. Dig Dis Sci. 2014;59(8):1743-53.
• 35. Brummer T, Zeiser R. The role of the MDM2/p53 axis in antitumor immune responses. Blood. 2024;143(26):2701-9.
• 36. Wang X, Zhu HQ, Lin SM, Xia BY, Xu B. RPN1: a pan-cancer biomarker and disulfidptosis regulator. Transl Cancer Res. 2024;13(5):2518-34.
• 37. Shearer BM, Sukov WR, Flynn HC, Knudson RA, Ketterling RP. Development of a dual-color, double fusion FISH assay to detect RPN1/EVI1 gene fusion associated with inv(3), t(3;3), and ins(3;3) in patients with myelodysplasia and acute myeloid leukemia. Am J Hematol. 2010;85(8):569-74.
• 38. Wang XX, Liu J, Tang YM, Hong L, Zeng Z, Tan GH. MicroRNA-638 inhibits cell proliferation by targeting suppress PIM1 expression in human osteosarcoma. Tumour Biol. 2017.
• 39. Huang B, Cai W, Wang Q, Liu F, Xu M, Zhang Y. Gankyrin Drives Malignant Transformation of Gastric Cancer and Alleviates Oxidative Stress via mTORC1 Activation. Oxid Med Cell Longev. 2018;2018:9480316.
• 40. Wu Y, Deng Y, Zhu J, Duan Y, Weng W, Wu X. Pim1 promotes cell proliferation and regulates glycolysis via interaction with MYC in ovarian cancer. Onco Targets Ther. 2018;11:6647-56.
• 41. Irshad K, Mohapatra SK, Srivastava C, Garg H, Mishra S, Dikshit B, et al. A combined gene signature of hypoxia and notch pathway in human glioblastoma and its prognostic relevance. PLoS One. 2015;10(3):e0118201.
• 42. Ameis HM, Drenckhan A, von Loga K, Escherich G, Wenke K, Izbicki JR, et al. PGK1 as predictor of CXCR4 expression, bone marrow metastases and survival in neuroblastoma. PLoS One. 2013;8(12):e83701.
• 43. Xu D, Aka JA, Wang R, Lin SX. 17beta-hydroxysteroid dehydrogenase type 5 is negatively correlated to apoptosis inhibitor GRP78 and tumor-secreted protein PGK1, and modulates breast cancer cell viability and proliferation. J Steroid Biochem Mol Biol. 2017;171:270-80.
• 44. Ai J, Huang H, Lv X, Tang Z, Chen M, Chen T, et al. FLNA and PGK1 are two potential markers for progression in hepatocellular carcinoma. Cell Physiol Biochem. 2011;27(3-4):207-16.
• 45. Chen J, Cao S, Situ B, Zhong J, Hu Y, Li S, et al. Metabolic reprogramming-based characterization of circulating tumor cells in prostate cancer. J Exp Clin Cancer Res. 2018;37(1):127.
• 46. He Y, Luo Y, Zhang D, Wang X, Zhang P, Li H, et al. PGK1-mediated cancer progression and drug resistance. Am J Cancer Res. 2019;9(11):2280-302.
• 47. Zhang X, Guan MX, Jiang QH, Li S, Zhang HY, Wu ZG, et al. NEAT1 knockdown suppresses endothelial cell proliferation and induces apoptosis by regulating miR638/AKT/mTOR signaling in atherosclerosis. Oncol Rep. 2020;44(1):115-25. 48. Jin HY, Gonzalez-Martin A, Miletic AV, Lai M, Knight S, Sabouri-Ghomi M, et al. Transfection of microRNA Mimics Should Be Used with Caution. Front Genet. 2015;6:340. 49. Manikkath J, Jishnu PV, Wich PR, Manikkath A, Radhakrishnan R. Nanoparticulate strategies for the delivery of miRNA mimics and inhibitors in anticancer therapy and its potential utility in oral submucous fibrosis. Nanomedicine (Lond). 2022;17(3):181-95.
• 50. Karatas OF, Wang J, Shao L, Ozen M, Zhang Y, Creighton CJ, et al. miR-33a is a tumor suppressor microRNA that is decreased in prostate cancer. Oncotarget. 2017;8(36):60243-56.
• 51. Song Y, Selak MA, Watson CT, Coutts C, Scherer PC, Panzer JA, et al. Mechanisms underlying metabolic and neural defects in zebrafish and human multiple acyl-CoA dehydrogenase deficiency (MADD). PLoS One. 2009;4(12):e8329.
• 52. Lee W, Kim J, Yun JM, Ohn T, Gong Q. MeCP2 regulates gene expression through recognition of H3K27me3. Nat Commun. 2020;11(1):3140.
• 53. Huang H, Zhao Y, Shang X, Ren H, Zhao Y, Liu X. CAIII expression in skeletal muscle is regulated by Ca(2+)-CaMKII-MEF2C signaling. Exp Cell Res. 2019;385(1):111672.
• 54. Boaretto F, Snijders D, Salvoro C, Spalletta A, Mostacciuolo ML, Collura M, et al. Diagnosis of Primary Ciliary Dyskinesia by a Targeted Next-Generation Sequencing Panel: Molecular and Clinical Findings in Italian Patients. J Mol Diagn. 2016;18(6):912-22.
• 55. 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.