Differences in plasma miRNA levels in inferior petrosal sinus samples of patients with ACTH-dependent Cushing’s syndrome

BACKGROUND: For the last decades microRNAs (miR) have proven themselves as novel biomarkers for various types of diseases. Identification of specific circulating microRNA panel that differ patient with Cushing’s disease (CD) and ectopic ACTH syndrome (EAS) could improve the diagnostic procedure.

AIM: to evaluate the differences in miR levels in plasma samples drained from inferior petrosal sinuses in patients with CD and EAS.

MATERIALS AND METHODS: single-center, case-control study: we enrolled 24 patients with ACTH-dependent Cushing’s syndrome (CS) requiring bilateral inferior petrosal sinus sampling (BIPSS).  Among them 12 subjects were confirmed as CD (males=2, females=10; median age 46,5 [IR 33,8;53,5]) and 12 as EAS (males=4, females=8, median age 54 [IR 38,75;60,75]). BIPSS was performed through a percutaneous bilateral approach. Once catheters were properly placed, blood samples were withdrawn simultaneously from each petrosal sinus and a peripheral vein. Plasma samples from both sinuses were centrifuged and then stored at -80 C. MiRNA isolation from plasma was carried out by an miRneasy Plasma/Serum Kit (Qiagen, Germany) on the automatic QIAcube station according to the manufacturer protocol. To prevent degradation, we added 1 unit of RiboLock Rnase Inhibitor (Thermo Fisher Scientific, USA) per 1 μL of RNA solution. The concentration of total RNA in the aqueous solution was evaluated on a NanoVue Plus spectrophotometer (GE Healthcare, USA). The libraries were prepared by the QIAseq miRNA Library Kit following the manufacturer standard protocols. MiR expression was then analyzed by sequencing on Illumina NextSeq 500 (Illumina, USA).

RESULTS: 108 miRNAs were differently expressed (p <0,05) in inferior petrosal sinus samples of patients with CD vs EAS. We divided these miRNAs into 3 groups based on the significance of the results. The first group consisted of samples with the highest levels of detected miR in both groups. Four miRNAs were included: miR-1203 was downregulated in CD vs EAS — 36.74 (p=0,013), and three other were upregulated in CD vs EAS: miR-383-3p 46.36 (p=0,01), miR-4290 6.84 (p=0,036), miR-6717-5p 4.49 (p=0,031). This miRs will be validated in larger cohorts using RT-qPCR.

CONCLUSION: Plasma miR levels differ in inferior petrosal samples taken from patients with CD vs EAS. These miRs need to be validated by different methods and in peripheral plasma samples in order to be used as potentially non-invasive biomarkers to differentiate ACTH-dependent CS.

1. Pivonello De Martino M, De Leo M, et al. Cushing’s disease: the burden of illness. Endocrine. 2016;56(1):10-18. doi: https://doi.org/10.1007/s12020-016-0984-8

2. Lonser R, Nieman L, Oldfield E. Cushing’s disease: pathobiology, diagnosis, and management. J Neurosurg. 2017;126(2):404-417. doi: https://doi.org/10.3171/2016.1.jns152119

3. Nieman LK, Biller BM, Findling JW, et al. The diagnosis of Cushing’s syndrome: an Endocrine Society Clinical Practice Guideline. Journal of Clinical Endocrinology and Metabolism. 2008;93:1526-1540. doi: https://doi.org/10.1210/jc.2008-0125

4. Sharma ST, Nieman LK, Feelders RA. Cushing’s syndrome: Epidemiology and developments in disease management. Clin Epidemiol. 2015;7:281-293. doi: https://doi.org/10.2147/CLEP.S44336

5. Lacroix A, Feelders R, Stratakis C, Nieman L. Cushing’s syndrome. The Lancet. 2015;386(9996):913-927. doi: https://doi.org/10.1016/s0140-6736(14)61375-1

6. Young J, Haissaguerre M, Viera-Pinto O, et al. Management of endocrine disease: Cushing’s syndrome due to ectopic ACTH secretion: an expert operational opinion. Eur J Endocrinol. 2020;182(4):R29-R58. doi: https://doi.org/10.1530/EJE-19-0877

7. Ilias I, Torpy D, Pacak K, et al. Cushing’s Syndrome Due to Ectopic Corticotropin Secretion: Twenty Years’ Experience at the National Institutes of Health. The Journal of Clinical Endocrinology & Metabolism. 2005;90(8):4955-4962. doi: https://doi.org/10.1210/jc.2004-2527

8. Sitkin II, Malygina AA, Belaya ZE, et al. Inferior petrosal sinus sampling in differential diagnosis of ACTH-dependent hypercortisolism. Endocr Surg. 2018;12(2):89-95. (In Russ.). doi: https://doi.org/10.14341/serg9752

9. Belaya ZE, Rozhinskaya LY, Dragunova NV, et al. Metabolic complications of endogenous Cushing: patient selection for screening. Obesity and metabolism. 2013;10(1):26-31. (In Russ.). doi: https://doi.org/10.14341/2071-8713-5068

10. Melnichenko GA, Dedov II, Belaya ZE, et al. Cushing’s disease: the clinical features, diagnostics, differential diagnostics, and methods of treatment. Problems of Endocrinology. 2015;61(2):55-77. (In Russ.). doi: https://doi.org/10.14341/probl201561255-77

11. Barbot M, Trementino L, Zilio M, et al. Second-line tests in the differential diagnosis of ACTH-dependent Cushing’s syndrome. Pituitary. 2016;19(5):488-495. doi: https://doi.org/10.1007/s11102-016-0729-y

12. Reimondo G, Paccotti P, Minetto M, et al. The corticotrophinreleasing hormone test is the most reliable noninvasive method to differentiate pituitary from ectopic ACTH secretion in Cushing’s syndrome. Clin Endocrinol (Oxf ). 2003;58(6):718-724. doi: https://doi.org/10.1046/j.1365-2265.2003.01776.x

13. Frete C, Corcuff J-B, Kuhn E, et al. Non-invasive Diagnostic Strategy in ACTH-dependent Cushing’s Syndrome. J Clin Endocrinol Metab. 2020;105(10):3273-3284. doi: https://doi.org/10.1210/clinem/dgaa409

14. Zampetti B, Grossrubatscher E, Dalino Ciaramella P, et al. Bilateral inferior petrosal sinus sampling. Endocr Connect. 2016;5(4):R12-R25. doi: https://doi.org/10.1530/EC-16-0029

15. Belaia ZE, Rozhinskaia LI, Mel’nichenko GA, et al. The role of prolactin gradient and normalized ACTH/prolactin ratio in the improvement of sensitivity and specificity of selective blood sampling from inferior petrosal sinuses for differential diagnostics of ACTHdependent hypercorticism. Problems of Endocrinology. 2013;59(4):3-10. (In Russ.). doi: https://doi.org/10.14341/probl20135943-10

16. Losa M, Allora A, Panni P, et al. Bilateral inferior petrosal sinus sampling in adrenocorticotropin-dependent hypercortisolism: always, never, or sometimes?. J Endocrinol Invest. 2019;42(8):997-1000. doi:10.1007/s40618-019-1006-5

17. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136(2):215-233. doi: https://doi.org/10.1016/j.cell.2009.01.002

18. Belaya Z, Grebennikova T, Melnichenko G, et al. Effects of active acromegaly on bone mRNA and microRNA expression patterns. Eur J Endocrinol. 2018;178(4):353-364. doi: https://doi.org/10.1530/EJE-17-0772

19. Belaya ZE, Grebennikova TA, Melnichenko GA, et al. Effects of endogenous hypercortisolism on bone mRNA and microRNA expression in humans. Osteoporos Int. 2018;29(1):211-221. doi: https://doi.org/10.1007/s00198-017-4241-7

20. Lutsenko AS, Belaya ZE, Przhiyalkovskaya EG, et al. Expression of plasma microRNA in patients with acromegaly. Problems of Endocrinology. 2019;65(5):311-318. (In Russ.) doi: https://doi.org/10.14341/probl10263

21. Lutsenko AS, Belaya ZE, Przhiyalkovskaya EG, et al. MicroRNA: role in gh-secreting pituitary adenoma pathogenesis. Ann Russ Acad Med Sci. 2017;72(4):290-298. (In Russ.). doi: https://doi.org/10.15690/vramn856

22. Aushev VN. MikroRNK: malye molekuly s bol’shim znacheniem. Klinicheskaya onkogematologiya. 2015;8(1):1-12. (In Russ.).

23. Hanahan D, Weinberg R. Hallmarks of Cancer: The Next Generation. Cell. 2011;144(5):646-674. doi: https://doi.org/10.1016/j.cell.2011.02.013

24. Calin GA, Dumitru CD, Shimizu M, et al. Nonlinear partial differential equations and applications: Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci. 2002;99(24):15524-15529. doi: https://doi.org/10.1073/pnas.242606799

25. Pashaei E, Pashaei E, Ahmady M, et al. Meta-analysis of miRNA expression profiles for prostate cancer recurrence following radical prostatectomy. PLoS One. 2017;12(6):e0179543. doi: https://doi.org/10.1371/journal.pone.0179543

26. Adhami M, Haghdoost AA, Sadeghi B, et al. Candidate miRNAs in human breast cancer biomarkers: a systematic review. Breast Cancer. 2018;25:198-205. doi: https://doi.org/10.1007/s12282-017-0814-803

27. Pardini B, De Maria D, Francavilla A, et al. MicroRNAs as markers of progression in cervical cancer: a systematic review. BMC Cancer. 2018;18(1):696. doi: https://doi.org/10.1186/s12885-018-4590-4

28. Shao C, Yang F, Qin Z, et al. The value of miR-155 as a biomarker for the diagnosis and prognosis of lung cancer: a systematic review with meta-analysis. BMC Cancer. 2019;19(1):1103. doi: https://doi.org/10.1186/s12885-019-6297-6

29. Belaya Z, Khandaeva P, Nonn L, et al. Circulating Plasma microRNA to Differentiate Cushing’s Disease From Ectopic ACTH Syndrome. Front Endocrinol (Lausanne). 2020;11:331. doi: https://doi.org/10.3389/fendo.2020.00331

30. Belaya ZE, Malygina AA, Grebennikova TA, et al. Diagnostic value of salivary cortisol in 1-mg dexamethasone suppression test. Obe Metab. 2020;17(1):13-21. (In Russ.). doi: https://doi.org/10.14341/omet10117

31. Chang L, Zhou G, Soufan O, Xia J. miRNet 2.0: networkbased visual analytics for miRNA functional analysis and systems biology. Nucleic Acids Res. 2020;48(W1):W244-W251. doi: https://doi.org/10.1093/nar/gkaa467

32. Bottoni A, Zatelli MC, Ferracin M, et al. Identification of differentially expressed microRNAs by microarray: a possible role for microRNA genes in pituitary adenomas. J Cell Physiol. 2007;210(2):370-377. doi: https://doi.org/10.1002/jcp.208321

33. Bottoni A, Piccin D, Tagliati F, et al. miR-15a and miR-16-1 downregulation in pituitary adenomas. J Cell Physiol. 2005;204(1):280-285. doi: https://doi.org/10.1002/jcp.20282

34. Yang Z Zhang T, Wang Q, Gao H. Overexpression of microRNA-34a Attenuates Proliferation and Induces Apoptosis in Pituitary Adenoma Cells via SOX7. Molecular Therapy - Oncolytics. 2018;10:40-47. doi: https://doi.org/10.1016/j.omto.2018.07.001

35. Amaral F, Torres N, Saggioro F, et al. MicroRNAs Differentially Expressed in ACTH-Secreting Pituitary Tumors. The Journal of Clinical Endocrinology & Metabolism. 2009;94(1):320-323. doi: https://doi.org/10.1210/jc.2008-1451

36. Stilling G, Sun Z, Zhang S et al. MicroRNA expression in ACTHproducing pituitary tumors: up-regulation of microRNA-122 and -493 in pituitary carcinomas. Endocrine. 2010;38(1):67-75. doi: https://doi.org/10.1007/s12020-010-9346-0

37. Vetrivel S, Zhang R, Engel M, et al. Circulating microRNA Expression in Cushing’s Syndrome. Front Endocrinol (Lausanne). 2021;12. doi: https://doi.org/10.3389/fendo.2021.620012

38. Lu B, Liu G, Yu F, et al. MicroRNA-16/VEGFR2/p38/NF-κB signaling pathway regulates cell growth of human pituitary neoplasms. Oncol Rep. 2018. doi: https://doi.org/10.3892/or.2018.6227

39. Renjie W, Haiqian L. MiR-132, miR-15a and miR-16 synergistically inhibit pituitary tumor cell proliferation, invasion and migration by targeting Sox5. Cancer Lett. 2015;356(2):568-578. doi: https://doi.org/10.1016/j.canlet.2014.10.003

40. Garbicz F, Mehlich D, Rak B et al. Increased expression of the microRNA 106b~25 cluster and its host gene MCM7 in corticotroph pituitary adenomas is associated with tumor invasion and Crooke’s cell morphology. Pituitary. 2017;20(4):450-463. doi: https://doi.org/10.1007/s11102-017-0805-y

41. Hernández-Ramírez L, Stratakis C. Genetics of Cushing’s Syndrome. Endocrinol Metab Clin North Am. 2018;47(2):275-297. doi: https://doi.org/10.1016/j.ecl.2018.02.007

42. Albani A, Theodoropoulou M, Reincke M. Genetics of Cushing’s disease. Clin Endocrinol (Oxf ). 2018;88(1):3-12. doi: https://doi.org/10.1111/cen.13457

43. Yanar EA, Makazan N V., Orlova EM, Kareva MА. Genetic basis of Cushing’s disease in children and targeted therapeutic future perspectives. Problems of Endocrinology. 2020;66(6):39-49. (In Russ.). doi: https://doi.org/10.14341/probl12676

44. Neou M, Villa C, Armignacco R, et al. Pangenomic Classification of Pituitary Neuroendocrine Tumors. Cancer Cell. 2020;37(1):123-134. doi: https://doi.org/10.1016/j.ccell.2019.11.002

45. Gao C, Lu W, Lou W, et al. Long noncoding RNA HOXC13‐AS positively affects cell proliferation and invasion in nasopharyngeal carcinoma via modulating miR‐383‐3p/HMGA2 axis. J Cell Physiol. 2018;234(8):12809-12820. doi: https://doi.org/10.1002/jcp.27915

46. Zhao L, Gu H, Chang J et al. MicroRNA-383 Regulates the Apoptosis of Tumor Cells through Targeting Gadd45g. PLoS One. 2014;9(11):e110472. doi: https://doi.org/10.1371/journal.pone.0110472

47. Qian Y, Song W, Wu X et al. DLX6 Antisense RNA 1 Modulates Glucose Metabolism and Cell Growth in Gastric Cancer by Targeting microRNA-4290. Dig Dis Sci. 2020;66(2):460-473. doi: https://doi.org/10.1007/s10620-020-06223-4

48. Nishibeppu K, Komatsu S, Imamura T, et al. Plasma microRNA profiles: identification of miR-1229-3p as a novel chemoresistant and prognostic biomarker in gastric cancer. Sci Rep. 2020;10(1):3161. doi: https://doi.org/10.1038/s41598-020-59939-8

49. Zou W, Cao Y, Cheng K, et al. Downregulation of circ_0037655 impedes glioma formation and metastasis via the regulation of miR-1229-3p/ITGB8 axis. Open Life Sciences. 2021;16(1):442-454. doi: https://doi.org/10.1515/biol-2021-0048

50. Wang Y, Yin Y, Zhou H, Cao Y. miR639 is associated with advanced cancer stages and promotes proliferation and migration of nasopharyngeal carcinoma. Oncol Lett. 2018. doi: https://doi.org/10.3892/ol.2018.9512

51. Lei S, Shen F, Chen J, et al. MiR-639 promoted cell proliferation and cell cycle in human thyroid cancer by suppressing CDKN1A expression. Biomedicine & Pharmacotherapy. 2016;84:1834-1840. doi: https://doi.org/10.1016/j.biopha.2016.10.087

52. Xiao J, Liu Y, Wu F, et al. miR-639 Expression Is Silenced by DNMT3AMediated Hypermethylation and Functions as a Tumor Suppressor in Liver Cancer Cells. Molecular Therapy. 2020;28(2):587-598. doi: https://doi.org/10.1016/j.ymthe.2019.11.021

53. Hamada-Tsutsumi S, Naito Y, Sato S, et al. The antiviral effects of human microRNA miR-302c-3p against hepatitis B virus infection. Aliment Pharmacol Ther. 2019;49(8):1060-1070. doi: https://doi.org/10.1111/apt.15197

54. Oksuz Z, Serin M, Kaplan E, et al. Serum microRNAs; miR-30c5p, miR-223-3p, miR-302c-3p and miR-17-5p could be used as novel non-invasive biomarkers for HCV-positive cirrhosis and hepatocellular carcinoma. Mol Biol Rep. 2014;42(3):713-720. doi: https://doi.org/10.1007/s11033-014-3819-9

55. Moreno C. SOX4: The unappreciated oncogene. Semin Cancer Biol. 2020;67:57-64. doi: https://doi.org/10.1016/j.semcancer.2019.08.027