Ruksolitinib ile Engellenen Glioblastoma İnvazyonunda AnjiyomiR’lerin Ekspresyon Profili

Amaç: MikroRNA’lar (miR), insan genomunda gen ifadesinin düzenlenmesinde önemli rolü olan düzenleyicilerdir. Son yıllarda anjiyogenezde rol oynayan spesifik bir miR grubu tanımlanmış (anjiyo-miR) ve bazı anjiyo-miR’lerin gliomalarda etkin rol oynadıkları ortaya konmuştur. Bu çalışmada, glioblastoma hücrelerindeki anjiyo-miR’lerin ifade değerlerindeki değişiklikleri ve bu değişikliklerin invazyon ve tümör büyümesi ile ilişkisini araştırdık. Gereç ve Yöntem: Bu çalışmada, insan glioblastoma hücre hattı U-87 MG kullanılarak glioblastoma tümör sferoidleri elde edildi. Matrigel yöntemi ile tümör sferoidlerine 48 saat süresince 50 nM, 100 nM and 200 nM ruksolitinib uygulandı. Kırk sekiz saat tedaviden sonra glioblastoma tümör sferoidlerinde tümör hacmi ve invazyon oluşumu relatif yüzde tümör gelişimi ve relatif yüzde invazyon alanı ölçüldü. Aynı zamanda, niceliksel gerçek zamanlı polimeraz zincir reaksiyonu (qRT-PZR) analizi yapıldı ve miR ifade değerleri belirlendi. Farklı şekillerde ifade edilen miRNA’ların modelini görüntülemek için normalize edilen miRNA ifade değerleri kullanılarak heatmap ve volcano plot analizleri ile seçilen en önemli (importance features) miRNA’lar gösterildi. Bulgular: Tümör sferoidlerine 50 nM, 100 nM ve 200 nM ruksolitinib uygulamasının tümör hacmi ve invazyon üzerine etkisi değerlendirildiğinde, uygulanan her dozda anlamlı fark saptandı. Ancak 200 nM ruksolitinib dozunda tümör yayılımını engelleyici etkisinin en yüksek olduğu gözlendi. 200 nM ruksolitinib uygulaması ile qRT-PZR testi ile elde edilen miR ifade değerleri incelendiğinde 10 miR’nin ifade değerlerinin arttığı, 4 tanesinin ifade değerlerinin ise azaldığı belirlendi. Sonuç: Sonuç olarak anjiyo-miR ifade değerleri gliomaların prognostik sürecini daha iyi anlamamızı sağlayabilmeleri açısından önemlidirler. Çoklu susturma özellikleri sayesinde glioblastomalar için yeni terapötik hedefler ve prognostik biyobelirteçler olarak kullanılabilmesi açısından ileri çalışmalarla kliniğe katkı sağlayabilirler

The Expression Profiles of Angio-miRs in Glioblastomas Invasion Inhibited by Ruxolitinib

Aim: MicroRNAs (miR) have an essential role on the regulated gene expression in the human genome. In recent years, a specific miR group was called to angio-miRs due to their role in the angiogenesis, and recent study showed that they involved in the pathogenesis of gliomas. In this study, we investigated the changes in the expression profiles of angio-miRs in glioblastoma cells and identified relationship between these genes and invasion and tumor growth. Materials and Methods: In this study, glioblastoma tumor spheroids were obtained using the human glioblastoma cell line U-87 MG. 50 nM, 100 nM and 200 nM ruxolitinib were applied to tumor spheroids for 48 hours by using Matrigell method. Tumor volume and invasion formation relative % tumor growth and relative % invasion area were measured in glioblastoma tumor spheroids after 48 hours of treatment. At the same time, quantitative real-time polymerase chain reaction (qRT-PZR) analysis was performed and miR expression profiles were determined. The most important (importance features) miRNAs selected along with the heatmap and volcano plot analyzes were used to display the pattern of the differentially expressed miRs using normalized miR expression profiles. Results: When the effect of 50 nM, 100 nM and 200 nM ruxolitinib administration to tumor spheroids on tumor volume and invasion was evaluated, a significant difference was found at each dose applied. However, at the dose of 200 nM ruxolitinib, it was observed that the inhibitory effect of tumor invasion was the highest. When miR expression profiles obtained by qRT-PZR test with 200 nM ruxolitinib adminisration were evaluated, it was determined that the expression profiles of 10 miRs increased and the expression profiles of 4 miRs decreased. Conclusion: In conclusion, angio-miR expression profiles are important because they enable us to better understand the prognostic process of gliomas. Because of their multiple silencing properties, they may contribute to the clinic with further studies in terms of their use as new therapeutic targets and prognostic biomarkers for glioblastoma.

PDF

___

1. Salinas-Vera YM, Marchat LA, Gallardo-Rincón D, Ruiz-García E, Astudillo- De La Vega H, Echavarría-Zepeda R, et al. AngiomiRs: MicroRNAs driving angiogenesis in cancer (Review). Int J Mol Med. 2019;43:657-70.
2. Pang C, Guan Y, Zhao K, Chen L, Bao Y, Cui R, et al. Up-regulation of microRNA-15b correlates with unfavorable prognosis and malignant progression of human glioma. Int J Clin Exp Pathol. 2015;8:4943-52.
3. Gabriely G, Wurdinger T, Kesari S, Esau CC, Burchard J, Linsley PS, et al. MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators. Mol Cell Biol. 2008;28:5369-80.
4. Anindo MI, Yaqinuddin A. Insights into the potential use of microRNAs as biomarker in cancer. Int J Surg. 2012;10:443-9.
5. Cuddapah VA, Robel S, Watkins S, Sontheimer H. A neuroentric perspective on glioma invasion. Nat Rev Neurosci. 2014;15:455-65.
6. Jain RK, di Tomaso E, Duda DG, Loeffler JS, Sorensen AG, Batchelor TT. Angiogenesis in brain tumours. Nat Rev Neurosci. 2007;8:610-22.
7. Price SJ, Gillard JH. Imaging biomarkers of brain tumour margin and tumour invasion. Br J Radiol. 2011;84(Spec No 2):S159-67.
8. Batchelor TT, Reardon DA, de Groot JF, Wick W, Weller M. Antiangiogenic therapy for glioblastoma: current status and future prospects. Clin Cancer Res. 2014;20:5612-9. 9. Beyer S, Fleming J, Meng W, Singh R, Haque SJ, Chakravarti A. The Role of miRNAs in Angiogenesis, Invasion and Metabolism and Their Therapeutic Implications in Gliomas. Cancers (Basel). 2017;9:85.
10. Delen E, Doğanlar O. The Dose Dependent Effects of Ruxolitinib on the Invasion and Tumorigenesis in Gliomas Cells via Inhibition of Interferon Gamma-Depended JAK/STAT Signaling Pathway. J Korean Neurosurg Soc. 2020;63:444-54.
11. Delen E, Doganlar O, Doganlar ZB, Delen O. Inhibition of the Invasion of Human Glioblastoma U87 Cell Line by Ruxolitinib: A Molecular Player of miR-17 and miR-20a Regulating JAK/STAT Pathway. Turk Neurosurg. 2020;30:182-9
12. Serocki M, Bartoszewska S, Janaszak-Jasiecka A, Ochocka RJ, Collawn JF, Bartoszewski R. miRNAs regulate the HIF switch during hypoxia: a novel therapeutic target. Angiogenesis. 2018;21:183-202.
13. Caporali A, Emanueli C. MicroRNA regulation in angiogenesis. Vascul Pharmacol. 2011;55:79-86.
14. Berthois Y, Delfino C, Metellus P, Fina F, Nanni-Metellus I, Al Aswy H, et al. Differential expression of miR200a-3p and miR21 in grade II-III and grade IV gliomas: evidence that miR200a-3p is regulated by O⁶-methylguanine methyltransferase and promotes temozolomide responsiveness. Cancer Biol Ther. 2014;15:938-50.
15. Barbano R, Palumbo O, Pasculli B, Galasso M, Volinia S, D’Angelo V, et al. A miRNA signature for defining aggressive phenotype and prognosis in gliomas. PLoS One. 2014;9:e108950.
16. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402-8.
17. Ma L, Teruya-Feldstein J, Weinberg RA. Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature. 2007;449:682-8.
18. Iseki Y, Shibutani M, Maeda K, Nagahara H, Fukuoka T, Matsutani S, et al. MicroRNA-96 Promotes Tumor Invasion in Colorectal Cancer via RECK. Anticancer Res. 2018;38:2031-5.
19. Fang LL, Wang XH, Sun BF, Zhang XD, Zhu XH, Yu ZJ, et al. Expression, regulation and mechanism of action of the miR-17-92 cluster in tumor cells (Review). Int J Mol Med. 2017;40:1624-30.
20. Xia H, Qi Y, Ng SS, Chen X, Chen S, Fang M, et al. MicroRNA-15b regulates cell cycle progression by targeting cyclins in glioma cells. Biochem Biophys Res Commun. 2009;380:205-10.
21. Ivo D’Urso P, Fernando D’Urso O, Damiano Gianfreda C, Mezzolla V, Storelli C, Marsigliante S. miR-15b and miR-21 as Circulating Biomarkers for Diagnosis of Glioma. Curr Genomics. 2015;16:304-11.
22. Baraniskin A, Kuhnhenn J, Schlegel U, Maghnouj A, Zöllner H, Schmiegel W, et al. Identification of microRNAs in the cerebrospinal fluid as biomarker for the diagnosis of glioma. Neuro Oncol. 2012;14:29-33.
23. Chen LP, Zhang NN, Ren XQ, He J, Li Y. miR-103/miR-195/miR-15b Regulate SALL4 and Inhibit Proliferation and Migration in Glioma. Molecules. 2018;23:2938.
24. Chen L, Li C, Zhang R, Gao X, Qu X, Zhao M, et al. miR-17-92 cluster microRNAs confers tumorigenicity in multiple myeloma. Cancer Lett. 2011;309:62-70.
25. Hsu TI, Hsu CH, Lee KH, Lin JT, Chen CS, Chang KC, et al. MicroRNA- 18a is elevated in prostate cancer and promotes tumorigenesis through suppressing STK4 in vitro and in vivo. Oncogenesis. 2014;3:e99.
26. Wu W, Zhou X, Yu T, Bao Z, Zhi T, Jiang K, et al. The malignancy of miR- 18a in human glioblastoma via directly targeting CBX7. Am J Cancer Res. 2017;7:64-76.
27. Song Y, Wang P, Zhao W, Yao Y, Liu X, Ma J, et al. MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin. Exp Cell Res. 2014;324:54-64.
28. Jiang Y, Zhou J, Zhao J, Hou D, Zhang H, Li L, et al. MiR-18a-downregulated RORA inhibits the proliferation and tumorigenesis of glioma using the TNF- α-mediated NF-κB signaling pathway. EBioMedicine. 2020;52:102651.
29. Peng Y, Huang D, Ma K, Deng X, Shao Z. MiR-19a as a prognostic indicator for cancer patients: a meta-analysis. Biosci Rep. 2019;39:BSR20182370.
30. Balachandran AA, Larcher LM, Chen S, Veedu RN. Therapeutically Significant MicroRNAs in Primary and Metastatic Brain Malignancies. Cancers (Basel). 2020;12:2534.
31. Malzkorn B, Wolter M, Liesenberg F, Grzendowski M, Stühler K, Meyer HE, et al. Identification and functional characterization of microRNAs involved in the malignant progression of gliomas. Brain Pathol. 2010;20:539-50.
32. Valastyan S, Reinhardt F, Benaich N, Calogrias D, Szász AM, Wang ZC,e t al . A pleiotropically acting microRNA, miR-31, inhibits breast cancer metastasis. Cell. 2009;137:1032-46.
33. Luo LJ, Yang F, Ding JJ, Yan DL, Wang DD, Yang SJ, et al. MiR-31 inhibits migration and invasion by targeting SATB2 in triple negative breast cancer. Gene. 2016;594:47-58.
34. Liu X, Sempere LF, Ouyang H, Memoli VA, Andrew AS, Luo Y, et al. MicroRNA-31 functions as an oncogenic microRNA in mouse and human lung cancer cells by repressing specific tumor suppressors. J Clin Invest. 2010;120:1298-309.
35. Cottonham CL, Kaneko S, Xu L. miR-21 and miR-31 converge on TIAM1 to regulate migration and invasion of colon carcinoma cells. J Biol Chem. 2010;285:35293-302.
36. Hua D, Ding D, Han X, Zhang W, Zhao N, Foltz G, et al. uman miR-31 targets radixin and inhibits migration and invasion of glioma cells. Oncol Rep. 2012;27:700-6.
37. Sato N, Funayama N, Nagafuchi A, Yonemura S, Tsukita S, Tsukita S. A gene family consisting of ezrin, radixin and moesin. Its specific localization at actin filament/plasma membrane association sites. J Cell Sci. 1992;103:131- 43.
38. Wang S, Jiao B, Geng S, Song J, Liang Z, Lu S. Concomitant microRNA-31 downregulation and radixin upregulation predicts advanced tumor progression and unfavorable prognosis in patients with gliomas. J Neurol Sci. 2014;338:71-6.
39. Rajbhandari R, McFarland BC, Patel A, Gerigk M, Gray GK, Fehling SC, et al. Loss of tumor suppressive microRNA-31 enhances TRADD/NF-κB signaling in glioblastoma. Oncotarget. 2015;6:17805-16.
40. Haan S, Margue C, Engrand A, Rolvering C, Schmitz-Van de Leur H, Heinrich PC, et al. Dual role of the Jak1 FERM and kinase domains in cytokine receptor binding and in stimulation-dependent Jak activation. J Immunol. 2008;180:998-1007.
41. Mattiske S, Suetani RJ, Neilsen PM, Callen DF. The oncogenic role of miR- 155 in breast cancer. Cancer Epidemiol Biomarkers Prev. 2012;21:1236-43.
42. Babar IA, Cheng CJ, Booth CJ, Liang X, Weidhaas JB, Saltzman WM, et al. Nanoparticle-based therapy in an in vivo microRNA-155 (miR- 155)-dependent mouse model of lymphoma. Proc Natl Acad Sci U S A. 2012;109:E1695-704.
43. Mallardo M, Poltronieri P, D’Urso OF. Non-protein coding RNA biomarkers and differential expression in cancers: a review. J Exp Clin Cancer Res. 2008;27:19.
44. D’Urso PI, D’Urso OF, Storelli C, Mallardo M, Gianfreda CD, Montinaro A, et al. miR-155 is up-regulated in primary and secondary glioblastoma and promotes tumour growth by inhibiting GABA receptors. Int J Oncol. 2012;41:228-34.
45. Jung E, Alfonso J, Osswald M, Monyer H, Wick W, Winkler F. Emerging intersections between neuroscience and glioma biology. Nat Neurosci. 2019;22:1951-60.
46. Men D, Liang Y, Chen L. Decreased expression of microRNA-200b is an independent unfavorable prognostic factor for glioma patients. Cancer Epidemiol. 2014;38:152-6.
47. Peng B, Hu S, Jun Q, Luo D, Zhang X, Zhao H, et al. MicroRNA-200b targets CREB1 and suppresses cell growth in human malignant glioma. Mol Cell Biochem. 2013;379:51-8.
48. Liu Q, Tang H, Liu X, Liao Y, Li H, Zhao Z, et al. miR-200b as a prognostic factor targets multiple members of RAB family in glioma. Med Oncol. 2014;31:859.
49. Chang YC, Su CY, Chen MH, Chen WS, Chen CL, Hsiao M. Secretory RAB GTPase 3C modulates IL6-STAT3 pathway to promote colon cancer metastasis and is associated with poor prognosis. Mol Cancer. 2017;16:135.