DOI: https://doi.org/10.29296/24999490-2023-04-06

I.R. Gilyazova(1–3), D.D. Asadullina(1, 2), E.A. Ivanova(1), A.A. Izmailov(4), D.A. Kudlay(5, 6), G.R. Gilyazova(2), E.F. Galimova(2),
I.B. Ermakov(4), R.R. Rakhimov(4), E.V. Popova4, A.F. Nasretdinov(4), A.B. Sultanbaev(4), E.K. Khusnutdinova(1), V.N. Pavlov(2)
1-Institute of Biochemistry and Genetics, Ufa Scientific Center, RAS, Oktyabrya Ave., 71, Ufa, 450054, Russian Federation;
2-Bashkir State Medical University, Lenin Str., 3, Ufa, 450008, Russian Federation;
3-«Saint Petersburg State University» (Department of Biology), Universitetskaya Emb., 7–9, St. Petersburg, 199034, Russian Federation;
4-Republican Clinical Oncology Dispensary, Oktyabrya Ave., 73/1, Ufa, 450054, Russian Federation;
5-I.M. Sechenov First Moscow State Medical University, Ministry of Health of Russia (Sechenov University),
Trubetskaya Str., 8, p. 2, Moscow, 119991, Russian Federation;
6-State Research Centre Institute of Immunology FMBA of Russia, Kashirskoe Hw, 24, Moscow, 115522, Russian Federation

Despite significant advance in clear cell renal cell carcinoma treatment, immune checkpoint inhibitors (ICIs) still have limited therapeutic efficacy. Taking into account the resistance to immunotherapy, observed in malignant neoplasms, the search for predictive markers of response to ICI therapy in patients with clear cell renal cell carcinoma (ccRCC) is under active investigation. Recent scientific studies demonstrate that exosomal miRNAs are key modulators of tumor signaling and determinants of the tumor microenvironment. Dysregulation of miRNAs can affect the immunogenicity of ccRCCs and response to ICI therapy, making them attractive as predictive molecular genetic biomarkers and targets for potential therapeutic developments. The aim of the study was to evaluate the expression levels of exosomal miRNAs-424,-503,-885,-149 in ccRCC patients who received ICI therapy. Material and methods: The study included 42 patients from whom venous blood samples were taken before and after ICI therapy. Expression analysis was performed by quantitative real-time PCR. Results: For miRNA-424 statistically significant differences in expression levels in the comparison groups were demonstrated. It was shown that the expression level of microRNA-424 increased after therapy (M±SM 1.202±0.15) compared with the expression level before treatment with nivolumab (M±SM 0.63±0.17; p-value=0.03). Despite the fact that miRNA-424 and miRNA-503 are clustered, miRNA-503, like other examined miRNAs, did not show any differences in expression levels between the compared groups. Conclusion: miRNA-424 can be used to create a panel of molecular markers within other previously discovered markers to assess the effectiveness of ICI therapy. Despite the fact that this study is pilot and requires validation on larger samples, it confirms the possibility of using miRNAs as additional prognostic markers for ICI therapy.
renal cell carcinoma; ICI therapy; exosomal miRNAs; immune-related adverse events; PD-1/PD-L1; biomarkers

Список литературы: 
  1. Kaprin АD. Malignant neoplasms in Russia in 2021. М: МNIOI PА Gerzena. 2022; 1–239.
  2. Brahmer J.R., Drake C.G., Wollner I., Powderly J.D., Picus J., Sharfman W.H. Phase I Study of Single-Agent Anti–Programmed Death-1 (MDX-1106) in Refractory Solid Tumors: Safety, Clinical Activity, Pharmacodynamics, and Immunologic Correlates. J. of Clinical Oncology. 2010; 28: 3167–75. https://doi.org/10.1200/JCO.2009.26.7609.
  3. Menzies A.M., Long G.V. New combinations and immunotherapies for melanoma: latest evidence and clinical utility. Ther Adv Med Oncol. 2013; 5: 278–85. https://doi.org/10.1177/1758834013499637.
  4. Tung I., Sahu A. Immune Checkpoint Inhibitor in First-Line Treatment of Metastatic Renal Cell Carcinoma: A Review of Current Evidence and Future Directions. Front Oncol. 2021; 11. https://doi.org/10.3389/fonc.2021.707214.
  5. Xu W., Atkins M.B., McDermott D.F. Checkpoint inhibitor immunotherapy in kidney cancer. Nat Rev Urol. 2020; 17: 137–50. https://doi.org/10.1038/s41585-020-0282-3.
  6. Yáñez-Mó M., Siljander P.R.-M., Andreu Z., Bedina Zavec A., Borràs F.E., Buzas E.I. Biological properties of extracellular vesicles and their physiological functions. J. Extracell Vesicles. 2015; 4: 27066. https://doi.org/10.3402/jev.v4.27066.
  7. Robbins P.D., Morelli A.E. Regulation of immune responses by extracellular vesicles. Nat Rev Immunol. 2014; 14: 195–208. https://doi.org/10.1038/nri3622.
  8. Bartel D.P. MicroRNAs. Cell. 2004; 116: 281–97. https://doi.org/10.1016/S0092-8674(04)00045-5.
  9. Zhang J., Li S., Li L., Li M., Guo C., Yao J. Exosome and Exosomal MicroRNA: Trafficking, Sorting, and Function. Genomics Proteomics Bioinformatics. 2015; 13: 17–24. https://doi.org/10.1016/j.gpb.2015.02.001.
  10. Suzuki H.I., Katsura A., Matsuyama H., Miyazono K. MicroRNA regulons in tumor microenvironment. Oncogene. 2015; 34: 3085–94. https://doi.org/10.1038/onc.2014.254.
  11. Ivanova E., Asadullina D., Gilyazova G., Rakhimov R., Izmailov A., Pavlov V. Exosomal MicroRNA Levels Associated with Immune Checkpoint Inhibitor Therapy in Clear Cell Renal Cell Carcinoma. Biomedicines. 2023; 11: 801. https://doi.org/10.3390/biomedicines11030801.
  12. Bhat P., Leggatt G., Waterhouse N., Frazer I.H. Interferon-γ derived from cytotoxic lymphocytes directly enhances their motility and cytotoxicity. Cell Death Dis. 2017; 8: 2836. https://doi.org/10.1038/cddis.2017.67.
  13. Burmeister A.R., Marriott I. The Interleukin-10 Family of Cytokines and Their Role in the CNS. Front Cell. Neurosci. 2018; 12. https://doi.org/10.3389/fncel.2018.00458.
  14. Zhou Y., Yamamoto Y., Takeshita F., Yamamoto T., Xiao Z., Ochiya T. Delivery of miR-424-5p via Extracellular Vesicles Promotes the Apoptosis of MDA-MB-231 TNBC Cells in the Tumor Microenvironment. Int. J. Mol. Sci. 2021; 22: 844. https://doi.org/10.3390/ijms22020844.
  15. Zheng H., Zhan Y., Liu S., Lu J., Luo J., Feng J. The roles of tumor-derived exosomes in non-small cell lung cancer and their clinical implications. J. of Experimental & Clinical Cancer Research. 2018; 37: 226. https://doi.org/10.1186/s13046-018-0901-5.
  16. Xu K., Zhang C., Du T., Gabriel A.N.A., Wang X., Li X. Progress of exosomes in the diagnosis and treatment of lung cancer. Biomedicine & Pharmacotherapy. 2021; 134: 111111. https://doi.org/10.1016/j.biopha.2020.111111.
  17. Taverna S., Giallombardo M., Gil-Bazo I., Carreca A.P., Castiglia M., Chacártegui J. Exosomes isolation and characterization in serum is feasible in non-small cell lung cancer patients: critical analysis of evidence and potential role in clinical practice. Oncotarget. 2016; 7: 28748–60. https://doi.org/10.18632/oncotarget.7638.
  18. Zhang J., Li S., Li L., Li M., Guo C., Yao J. Exosome and Exosomal MicroRNA: Trafficking, Sorting, and Function. Genomics Proteomics Bioinformatics. 2015; 13: 17–24. https://doi.org/10.1016/j.gpb.2015.02.001.
  19. Forrest A.R.R., Kanamori-Katayama M., Tomaru Y., Lassmann T., Ninomiya N., Takahashi Y. Induction of microRNAs, mir-155, mir-222, mir-424 and mir-503, promotes monocytic differentiation through combinatorial regulation. Leukemia. 2010; 24: 460–6. https://doi.org/10.1038/leu.2009.246.
  20. Finnerty J.R., Wang W.-X., Hébert S.S., Wilfred B.R., Mao G., Nelson P.T. The miR-15/107 Group of MicroRNA Genes: Evolutionary Biology, Cellular Functions, and Roles in Human Diseases. J. Mol. Biol. 2010; 402: 491–509. https://doi.org/10.1016/j.jmb.2010.07.051.
  21. Wang F., Liang R., Tandon N., Matthews E.R., Shrestha S., Yang J. H19X-encoded miR-424(322)/-503 cluster: emerging roles in cell differentiation, proliferation, plasticity and metabolism. Cellular and Molecular Life Sciences. 2019; 76: 903–20. https://doi.org/10.1007/s00018-018-2971-0.
  22. Xuan J., Liu Y., Zeng X., Wang H. Sequence Requirements for miR-424-5p Regulating and Function in Cancers. Int J. Mol. Sci. 2022; 23 (7): 4037. DOI: 10.3390/ijms23074037.
  23. Xu S., Tao Z., Hai B., Liang H., Shi Y., Wang T. miR-424(322) reverses chemoresistance via T-cell immune response activation by blocking the PD-L1 immune checkpoint. Nat Commun. 2016; 7: 11406. https://doi.org/10.1038/ncomms11406.
  24. Zhao X., Yuan C., Wangmo D., Subramanian S. Tumor-Secreted Extracellular Vesicles Regulate T-Cell Costimulation and Can Be Manipulated To Induce Tumor-Specific T-Cell Responses. Gastroenterology. 2021; 161: 560–74. e11. https://doi.org/10.1053/j.gastro.2021.04.036.
  25. Zhou Y., Yamamoto Y., Takeshita F., Yamamoto T., Xiao Z., Ochiya T. Delivery of miR-424-5p via Extracellular Vesicles Promotes the Apoptosis of MDA-MB-231 TNBC Cells in the Tumor Microenvironment. Int J. Mol. Sci. 2021; 22: 844. https://doi.org/10.3390/ijms22020844.
  26. Nowak M., Klink M., Glowacka E., Sulowska Z., Kulig A., Szpakowski M. Production of Cytokines During Interaction of Peripheral Blood Mononuclear Cells with Autologous Ovarian Cancer Cells or Benign Ovarian Tumour Cells. Scand J. Immunol. 2010; 71: 91–8. https://doi.org/10.1111/j.1365-3083.2009.02350.x.
  27. Chen B., Duan L., Yin G., Tan J., Jiang X. Simultaneously expressed miR-424 and miR-381 synergistically suppress the proliferation and survival of renal cancer cells-Cdc2 activity is up-regulated by targeting WEE1. Clinics. 2013; 68: 825–33. https://doi.org/10.6061/clinics/2013(06)17.
  28. Liu J., Gu Z., Tang Y., Hao J., Zhang C., Yang X. Tumour-suppressive microRNA-424-5p directly targets CCNE1 as potential prognostic markers in epithelial ovarian cancer. Cell Cycle. 2018; 17: 309–18. https://doi.org/10.1080/15384101.2017.1407894.
  29. Zhao C., Zhao F., Chen H., Liu Y., Su J. MicroRNA-424-5p inhibits the proliferation, migration, and invasion of nasopharyngeal carcinoma cells by decreasing AKT3 expression. Brazilian J. of Medical and Biological Research. 2020; 53. https://doi.org/10.1590/1414-431x20209029.
  30. Wu J., Yang B., Zhang Y., Feng X., He B., Xie H. miR-424-5p represses the metastasis and invasion of intrahepatic cholangiocarcinoma by targeting ARK5. Int. J. Biol. Sci. 2019; 15: 1591–9. https://doi.org/10.7150/ijbs.34113.
  31. Dong J., Wang Q., Li L., Xiao-jin Z. Upregulation of Long Non-Coding RNA Small Nucleolar RNA Host Gene 12 Contributes to Cell Growth and Invasion in Cervical Cancer by Acting as a Sponge for MiR-424-5p. Cellular Physiology and Biochemistry. 2018; 45: 2086–94. https://doi.org/10.1159/000488045.
  32. Jin C., Li M., Ouyang Y., Tan Z., Jiang Y. MiR-424 functions as a tumor suppressor in glioma cells and is down-regulated by DNA methylation. J. Neurooncol. 2017; 133: 247–55. https://doi.org/10.1007/s11060-017-2438-4.
  33. Li T., Li Y., Gan Y., Tian R., Wu Q., Shu G. Methylation-mediated repression of MiR-424/503 cluster promotes proliferation and migration of ovarian cancer cells through targeting the hub gene KIF23. Cell Cycle. 2019; 18: 1601–18. https://doi.org/10.1080/15384101.2019.1624112.
  34. Ghonbalani Z.N., Shahmohamadnejad S., Pasalar P., Khalili E. Hypermethylated miR-424 in Colorectal Cancer Subsequently Upregulates VEGF. J. Gastrointest Cancer. 2022; 53: 380–6. https://doi.org/10.1007/s12029-021-00614-0.
  35. Gowrishankar B., Ibragimova I., Zhou Y., Slifker M.J., Devarajan K., Al-Saleem T. MicroRNA expression signatures of stage, grade, and progression in clear cell RCC. Cancer Biol Ther. 2014; 15: 329–41. https://doi.org/10.4161/cbt.27314.
  36. Li Y., Liu J., Hu W., Zhang Y., Sang J., Li H. <p>miR-424-5p Promotes Proliferation, Migration and Invasion of Laryngeal Squamous Cell Carcinoma. Onco Targets Ther. 2019; 12: 10441–53. https://doi.org/10.2147/OTT.S224325.
  37. Dai W., Zhou J., Wang H., Zhang M., Yang X., Song W. miR-424-5p promotes the proliferation and metastasis of colorectal cancer by directly targeting SCN4B. Pathol Res Pract. 2020; 216: 152731. https://doi.org/10.1016/j.prp.2019.152731.
  38. Moynihan M.J., Sullivan T.B., Burks E., Schober J., Calabrese M., Fredrick A. MicroRNA profile in stage I clear cell renal cell carcinoma predicts progression to metastatic disease. Urologic Oncology: Seminars and Original Investigations. 2020; 38: 799. https://doi.org/10.1016/j.urolonc.2020.05.006.
  39. Kalantzakos T.J., Sullivan T.B., Gloria T., Canes D., Moinzadeh A., Rieger-Christ K.M. MiRNA-424-5p Suppresses Proliferation, Migration, and Invasion of Clear Cell Renal Cell Carcinoma and Attenuates Expression of O-GlcNAc-Transferase. Cancers (Basel). 2021; 13: 5160. https://doi.org/10.3390/cancers13205160.
  40. Peng X.-X., Yu R., Wu X., Wu S.-Y., Pi C., Chen Z.-H. Correlation of plasma exosomal microRNAs with the efficacy of immunotherapy in EGFR/ALK wild-type advanced non-small cell lung cancer. J. Immunother Cancer. 2020; 8: 000376. https://doi.org/10.1136/jitc-2019-000376.
  41. Halvorsen A.R., Sandhu V., Sprauten M., Flote V.G., Kure E.H., Brustugun O.T. Circulating microRNAs associated with prolonged overall survival in lung cancer patients treated with nivolumab. Acta Oncol (Madr). 2018; 57: 1225–31. https://doi.org/10.1080/0284186X.2018.1465585.
  42. Boeri M., Milione M., Proto C., Signorelli D., Lo Russo G., Galeone C. Circulating miRNAs and PD-L1 Tumor Expression Are Associated with Survival in Advanced NSCLC Patients Treated with Immunotherapy: a Prospective Study. Clinical Cancer Research. 2019; 25: 2166–73. https://doi.org/10.1158/1078-0432.CCR-18-1981.
  43. Pantano F., Zalfa F., Iuliani M., Simonetti S., Manca P., Napolitano A. Large-Scale Profiling of Extracellular Vesicles Identified miR-625-5p as a Novel Biomarker of Immunotherapy Response in Advanced Non-Small-Cell Lung Cancer Patients. Cancers (Basel). 2022; 14: 2435. https://doi.org/10.3390/cancers14102435.
  44. Xiao Q.-Z., Zhu L.-J., Fu Z.-Y., Guo X.-R., Chi X. Obesity related microRNA424 is regulated by TNF&amp;alpha; in adipocytes. Mol. Med Rep. 2020; 23: 1. https://doi.org/10.3892/mmr.2020.11659.
  45. Gramantieri L., Giovannini C., Piscaglia F., Fornari F. MicroRNAs as Modulators of Tumor Metabolism, Microenvironment, and Immune Response in Hepatocellular Carcinoma. J. Hepatocell Carcinoma. 2021; 8: 369–85. https://doi.org/10.2147/JHC.S268292.
  46. Gui J., Tian Y., Wen X., Zhang W., Zhang P., Gao J. Serum microRNA characterization identifies miR – 885 – 5p as a potential marker for detecting liver pathologies. Clin Sci. 2011; 120: 183–93. https://doi.org/10.1042/CS20100297.
  47. Raitoharju E., Seppälä I., Lyytikäinen L.-P., Viikari J., Ala-Korpela M., Soininen P. Blood hsa-miR-122-5p and hsa-miR-885-5p levels associate with fatty liver and related lipoprotein metabolism. The Young Finns Study. Sci Rep. 2016; 6: 38262. https://doi.org/10.1038/srep38262.
  48. Liu Y., Bao Z., Tian W., Huang G. miR8855p suppresses osteosarcoma proliferation, migration and invasion through regulation of βcatenin. Oncol Lett. 2018. https://doi.org/10.3892/ol.2018.9768.
  49. Jin X., Wang Z., Pang W., Zhou J., Liang Y., Yang J. Upregulated hsa_circ_0004458 Contributes to Progression of Papillary Thyroid Carcinoma by Inhibition of miR-885-5p and Activation of RAC1. Medical Science Monitor. 2018; 24: 5488–500. https://doi.org/10.12659/MSM.911095.
  50. Zhang Z., Yin J., Yang J., Shen W., Zhang C., Mou W. miR-885-5p suppresses hepatocellular carcinoma metastasis and inhibits Wnt/β-catenin signaling pathway. Oncotarget. 2016; 7: 75038–51. https://doi.org/10.18632/oncotarget.12602.
  51. Yao D., Xia S., Jin C., Zhao W., Lan W., Liu Z. Feedback activation of GATA1/miR-885-5p/PLIN3 pathway decreases sunitinib sensitivity in clear cell renal cell carcinoma. Cell Cycle. 2020; 19: 2195–206. https://doi.org/10.1080/15384101.2020.1801189.
  52. Lu X., Jing L., Liu S., Wang H., Chen B. miR-149-3p Is a Potential Prognosis Biomarker and Correlated with Immune Infiltrates in Uterine Corpus Endometrial Carcinoma. Int. J. Endocrinol. 2022; 2022: 1–15. https://doi.org/10.1155/2022/5006123.
  53. Zhang M., Gao D., Shi Y., Wang Y., Joshi R., Yu Q. miR-149-3p reverses CD8 + T-cell exhaustion by reducing inhibitory receptors and promoting cytokine secretion in breast cancer cells. Open Biol. 2019; 9: 190061. https://doi.org/10.1098/rsob.190061.
  54. Jin L., li Y., Liu J., Yang S., Gui Y., Mao X. Tumor suppressor miR-149-5p is associated with cellular migration, proliferation and apoptosis in renal cell carcinoma. Mol Med Rep. 2016; 13: 5386–92. https://doi.org/10.3892/mmr.2016.5205.
  55. Okato A., Arai T., Yamada Y., Sugawara S., Koshizuka K., Fujimura L. Dual Strands of Pre-miR-149 Inhibit Cancer Cell Migration and Invasion through Targeting FOXM1 in Renal Cell Carcinoma. Int. J. Mol. Sci. 2017; 18: 1969. https://doi.org/10.3390/ijms18091969.
  56. Ke Y., Zhao W., Xiong J., Cao R. miR-149 Inhibits Non-Small-Cell Lung Cancer Cells EMT by Targeting FOXM1. Biochem Res Int. 2013; 2013: 1–8. https://doi.org/10.1155/2013/506731.
  57. She X., Yu Z., Cui Y., Lei Q., Wang Z., Xu G. miR-128 and miR-149 enhance the chemosensitivity of temozolomide by Rap1B-mediated cytoskeletal remodeling in glioblastoma. Oncol Rep. 2014; 32: 957–64. https://doi.org/10.3892/or.2014.3318.
  58. Min S., Liang X., Zhang M., Zhang Y., Mei S., Liu J. Multiple Tumor-Associated MicroRNAs Modulate the Survival and Longevity of Dendritic Cells by Targeting YWHAZ and Bcl2 Signaling Pathways. The J. of Immunology. 2013; 190: 2437–46. https://doi.org/10.4049/jimmunol.1202282.
  59. Xiao F., Zhang W., Chen L., Chen F., Xie H., Xing C. MicroRNA-503 inhibits the G1/S transition by downregulating cyclin D3 and E2F3 in hepatocellular carcinoma. J. Transl. Med. 2013; 11: 195. https://doi.org/10.1186/1479-5876-11-195.
  60. Li B., Liu L., Li X., Wu L. miR-503 suppresses metastasis of hepatocellular carcinoma cell by targeting PRMT1. Biochem Biophys Res Commun. 2015; 464: 982–7. https://doi.org/10.1016/j.bbrc.2015.06.169.
  61. Yang Y., Liu L., Zhang Y., Guan H., Wu J., Zhu X. MiR-503 targets PI3K p85 and IKK-β and suppresses progression of non-small cell lung cancer. Int. J. Cancer. 2014; 135: 1531–42. https://doi.org/10.1002/ijc.28799.
  62. Qiu T., Zhou L., Wang T., Xu J., Wang J., Chen W. miR-503 regulates the resistance of non-small cell lung cancer cells to cisplatin by targeting Bcl-2. Int. J. Mol. Med. 2013; 32: 593–8. https://doi.org/10.3892/ijmm.2013.1439.