İnsan trofoblast hücre hattinda sialik asit bağlayici reseptörlerin (siglecs) ekspresyonu

Yıl 2024, Cilt: 17 Sayı: 2, 195 - 203, 01.04.2024

Nazlı Çil , İ.veysel Fenkci , Gülçin Abban Mete , Doğukan Mutlu , Cihan Kabukçu , Ümit Çabuş

https://doi.org/10.31362/patd.1346146

Öz

Amaç: Siglecler olarak bilinen hücre yüzeyi proteinleri, sialik asit için özel bir afinite sergilemektedir. Öncelikle bağışıklık hücrelerinin yüzeyinde bulunan Siglec'ler, I-tipi lektinler adı verilen lektin alt kümesine aittir. Siglecler maternal-fetal immün toleransta önemli rollere sahiptir. Bu çalışmada İnsan Villöz Trofoblast (HVT) hücre hatlarında Siglec tiplerinin belirlenmesi amaçlanmıştır.
Gereç ve yöntem: HVT hücre hattından total RNA ekstrakte edildi, cDNA sentezlendi ve Siglec -3, -5, -6, -7, -8, -9, -10, -11 ve -16 gen ekspresyonu Gerçek Zamanlı Kantitatif Ters Transkripsiyon Polimeraz Zincir Reaksiyonu (qRT-PCR) ile analiz edildi. Ek olarak, Siglec seviyelerini belirlemek için immünohistokimyasal boyamalar yapılmıştır. Siglec-6 ve Siglec-9' a karşı immünoreaktivite kahverengi rengin yoğunluğuna göre ayrı ayrı değerlendirilmiştir.
Bulgular: HVT hücre hattından ekstrakte edilen RNA kullanılarak Siglec genlerinin qRT-PCR ile ekspresyon seviyeleri ile erime eğrisi analizleri yapıldı. Siglec -3, -5, -6, -7, -9, -10, -11 ve -16 genlerinin HVT hücre hattında ifade edildiği bulundu. Farklı olarak, Siglec-8 sonuçları 40. döngüden sonra tespit edilemedi ve bu da negatif bir sonuç olarak kabul edildi. HVT hücre hattının immünositokimyasal incelemesinde Siglec-6 ekspresyonunun sitoplazmada orta düzeyde olduğu ortaya çıkmıştır (Skor:2). Siglec-9 ekspresyonu sitoplazmada belirgindi (Skor:3).
Sonuç: Sonuçlar Siglec-6 ve Siglec-9'un HVT hücre hattında diğer Siglec proteinlerinden daha fazla ifade edildiğini göstermiştir. İmmünositokimya sonuçları da bu bulguları desteklemektedir. Çalışmamız insan trofoblast hücrelerinde Siglec-9 ekspresyonunu gösteren ilk çalışmadır.

Anahtar Kelimeler

Siglecs, insan villöz trafoblast, sialik asit

Proje Numarası

2022HZDP003

Expression of sialic acid binding receptors (siglecs) in human trophoblast cell line

Öz

Purpose: Cell surface proteins known as Siglecs exhibit a specific affinity for sialic acid. Primarily located on the surface of immune cells, Siglecs belong to the subset of lectins called I-type lectins. Siglecs have important roles in maternal-fetal immune tolerance. We aimed to analyze the expression of Siglecs on Human Villous Trophoblasts (HVT) cells.
Materials and methods: Total RNA was extracted from the HVT cell line, cDNA was synthesized, and real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed the determine the changes in Siglec -3, -5, -6, -7, -8, -9, -10, -11, and -16 mRNA levels. In addition, Siglec levels were assessed by using immunohistochemical staining. Immunoreactivity against Siglec-6 and Siglec-9 was evaluated separately according to the intensity of brown color.
Results: Expression levels of Siglec genes by qRT-PCR and melting curve analyses were performed using RNA extracted from the HVT cell line. Siglec -3, -5, -6, -7, -9, -10, -11, and -16 genes were found to be expressed in the HVT cell line. Differently, Siglec-8 results were undetected after cycle 40, which was considered a negative result. The immunocytochemical examination of the HVT cell line revealed that Siglec-6 expression was moderate in the cytoplasm (Score:2). Siglec-9 expression was prominent in the cytoplasm (Score:3).
Conclusion: The results showed that Siglec-6 and Siglec-9 were expressed more than other Siglec proteins in the human trophoblast cells. Immunocytochemistry results also support these findings. Our study is the first to show Siglec-9 expression in human trophoblast cell.

Anahtar Kelimeler

Siglecs, human villous trophoblasts, sialic acid

Destekleyen Kurum

This work was supported by The Pamukkale University Scientific Research Projects Coordination Unit

Proje Numarası

2022HZDP003

Kaynakça

• 1. World Health Organization. Recommendations. In: Vayena E, Rowe PJ, Griffin PD. Current practices and controversies in assisted reproduction: report of a meeting on "Medical, Ethical and Social Aspects of Assisted Reproduction" held at WHO Headquarters in Geneva, Switzerland: World Health Organization 2002;381-396.
• 2. Vander Borgh M, Wyns C. Fertility and infertility: definition and epidemiology. Clin Biochem 2018;62:2-10. https://doi.org/10.1016/j.clinbiochem.2018.03.012
• 3. Stephen EH. Projections of impaired fecundity among women in the United States: 1995 to 2020. Fertil Steril 1996;66:205-209. https://doi.org/10.1016/s0015-0282(16)58439-8
• 4. Wilkes S, Chinn DJ, Murdoch A, Rubin G. Epidemiology and management of infertility: a population-based study in UK primary care. Fam Pract 2009;26:269-274. https://doi.org/10.1093/fampra/cmp029
• 5. Dekel N, Gnainsky Y, Granot I, Mor G. Inflammation and implantation. Am J Reprod Immunol 2010;63:17-21. https://doi.org/10.1111/j.1600-0897.2009.00792.x
• 6. Horcajadas JA, Pellicer A, Simón C. Wide genomic analysis of human endometrial receptivity: new times, new opportunities. Hum Reprod Update 2007;13:77-86. https://doi.org/10.1093/humupd/dml046
• 7. Salamonsen LA, Nie G, Findlay JK. Newly identified endometrial genes of importance for implantation. J Reprod Immunol 2002;53:215-225. https://doi.org/10.1016/s0165-0378(01)00087-0
• 8. Varki A. Glycan-based interactions involving vertebrate sialic-acid-recognizing proteins. Nature 2007;446:1023-1029. https://doi.org/10.1038/nature05816
• 9. Bronikowska I, Świętochowska E, Oleksiak M, Czecior E. Sialic acids in squamous cell carcinoma of the head and neck. Postepy Hig Med Dosw (Online) 2016;70:1300-1308. https://doi.org/10.5604/17322693.1227410
• 10. RodrÍguez E, Schetters STT, van Kooyk Y. The tumour glyco-code as a novel immune checkpoint for immunotherapy. Nat Rev Immunol 2018;18:204-211. https://doi.org/10.1038/nri.2018.3
• 11. Borzym Kluczyk M, Radziejewska I, Cechowska Pasko M, Darewicz B. Reduced expression of E-cadherin and increased sialylation level in clear cell renal cell carcinoma. Acta Biochim Pol 2017;64:465-470. https://doi.org/10.18388/abp.2015_1215
• 12. Pihikova D, Kasak P, Kubanikova P, Sokol R, Tkac J. Aberrant sialylation of a prostate-specific antigen: electrochemical label-free glycoprofiling in prostate cancer serum samples. Anal Chim Acta 2016;31;934:72-79. https://doi.org/10.1016/j.aca.2016.06.043
• 13. Zhang Z, Wuhrer M, Holst S. Serum sialylation changes in cancer. Glycoconj J 2018;35:139-160. https://doi.org/10.1007/s10719-018-9820-0
• 14. Adams OJ, Stanczak MA, von Gunten S, Läubli H. Targeting sialic acid-Siglec interactions to reverse immune suppression in cancer. Glycobiology 2018;28:640-647. https://doi.org/10.1093/glycob/cwx108
• 15. Nan X, Carubelli I, Stamatos NM. Sialidase expression in activated human T lymphocytes influences production of IFN-gamma. J Leukoc Biol 2007;81:284-296. https://doi.org/10.1189/jlb.1105692
• 16. Li N, Zhang W, Wan T, et al. Cloning and characterization of Siglec-10, a novel sialic acid binding member of the Ig superfamily, from human dendritic cells. J Biol Chem 2001;276:28106-28112. https://doi.org/10.1074/jbc.M100467200
• 17. Higuchi H, Shoji T, Iijima S, Nishijima K. Constitutively expressed Siglec-9 inhibits LPS-induced CCR7, but enhances IL-4-induced CD200R expression in human macrophages. Biosci Biotechnol Biochem 2016;80:1141-1148. https://doi.org/10.1080/09168451.2016.1146070
• 18. Lock K, Zhang J, Lu J, Lee SH, Crocker PR. Expression of CD33-related siglecs on human mononuclear phagocytes, monocyte-derived dendritic cells and plasmacytoid dendritic cells. Immunobiology 2004;209:199-207. https://doi.org/10.1016/j.imbio.2004.04.007
• 19. Büll C, Collado Camps E, Kers Rebel ED, et al. Metabolic sialic acid blockade lowers the activation threshold of moDCs for TLR stimulation. Immunol Cell Biol 2017;95:408-415. https://doi.org/10.1038/icb.2016.105
• 20. De Saint Jean A, Lucht F, Bourlet T, Delézay O. Transforming growth factor beta 1 up-regulates CD169 (sialoadhesin) expression on monocyte-derived dendritic cells: role in HIV sexual transmission. AIDS 2014;28:2375-2380. https://doi.org/10.1097/QAD.0000000000000431
• 21. Angata T, Varki A. Siglec-7: a sialic acid-binding lectin of the immunoglobulin superfamily. Glycobiology 2000;10:431-438. https://doi.org/10.1093/glycob/10.4.431
• 22. Angata T, Varki A. Cloning, characterization, and phylogenetic analysis of siglec-9, a new member of the CD33-related group of siglecs. Evidence for co-evolution with sialic acid synthesis pathways. J Biol Chem 2000;275:22127-22135. https://doi.org/10.1074/jbc.M002775200
• 23. Zhang JQ, Nicoll G, Jones C, Crocker PR. Siglec-9, a novel sialic acid binding member of the immunoglobulin superfamily expressed broadly on human blood leukocytes. J Biol Chem 2000;275:22121-22126. https://doi.org/10.1074/jbc.M002788200
• 24. Avril T, Attrill H, Zhang J, Raper A, Crocker PR. Negative regulation of leucocyte functions by CD33-related siglecs. Biochem Soc Trans 2006;34:1024-1027. https://doi.org/10.1042/BST0341024
• 25. Poe JC, Hasegawa M, Tedder TF. CD19, CD21, and CD22: multifaceted response regulators of B lymphocyte signal transduction. Int Rev Immunol 2001;20:739-762. https://doi.org/10.3109/08830180109045588
• 26. Ali SR, Fong JJ, Carlin AF, et al. Siglec-5 and Siglec-14 are polymorphic paired receptors that modulate neutrophil and amnion signaling responses to group B Streptococcus. J Exp Med 2014;211:1231-1242. https://doi.org/10.1084/jem.20131853
• 27. Roseman DS, Baenziger JU. The Man/GalNAc-4-SO4-receptor: relating specificity to function. Methods Enzymol 2003;363:121-133. https://doi.org/10.1016/S0076-6879(03)01048-6
• 28. Mutlu D, Cakir C, Ozturk M, Arslan S. Anticancer and apoptotic effects of a polysaccharide extract isolated from lactarius chrysorrheus Fr. in HepG2 and PANC-1 cell lines. Archives of Biological Sciences 2022;74:315-324. https://doi.org/10.2298/ABS220803030M
• 29. Kim M, Suh DH, Choi JY, et al. Mutually exclusive antiproliferative effect of cell line-specific HOX inhibition in epithelial ovarian cancer cell lines: SKOV-3 vs RMUG-S. J Cell Mol Med 2020;24:3246-3251. https://doi.org/10.1111/jcmm.14993
• 30. Yang J, Zhou Y, Xie S, et al. Metformin induces Ferroptosis by inhibiting UFMylation of SLC7A11 in breast cancer. J Exp Clin Cancer Res 2021;40:206. https://doi.org/10.1186/s13046-021-02012-7
• 31. Mengerink KJ, Vacquier VD. Glycobiology of sperm-egg interactions in deuterostomes. Glycobiology 2001;11:37-43. https://doi.org/10.1093/glycob/11.4.37r
• 32. Brayman M, Thathiah A, Carson DD. MUC1: a multifunctional cell surface component of reproductive tissue epithelia. Reprod Biol Endocrinol 2004;2:4. https://doi.org/10.1186/1477-7827-2-4
• 33. Chatterji U, Sen AK, Schauer R, Chowdhury M. Paracrine effects of a uterine agglutinin are mediated via the sialic acids present in the rat uterine endometrium. Mol Cell Biochem 2000;215:47-55. https://doi.org/10.1023/a:1026582715752
• 34. Velásquez JG, Canovas S, Barajas P, et al. Role of sialic acid in bovine sperm-zona pellucida binding. Mol Reprod Dev 2007;74:617-628. https://doi.org/10.1002/mrd.20619
• 35. Schwarzkopf M, Knobeloch KP, Rohde E, et al. Sialylation is essential for early development in mice. Proc Natl Acad Sci U S A 2002;99:5267-5270. https://doi.org/10.1073/pnas.072066199
• 36. Tecle E, Reynoso HS, Wang R, Gagneux P. The female reproductive tract contains multiple innate sialic acid-binding immunoglobulin-like lectins (Siglecs) that facilitate sperm survival. J Biol Chem 2019;294:11910-11919. https://doi.org/10.1074/jbc.RA119.008729
• 37. Razi N, Varki A. Cryptic sialic acid binding lectins on human blood leukocytes can be unmasked by sialidase treatment or cellular activation. Glycobiology 1999;9:1225-1234. https://doi.org/10.1093/glycob/9.11.1225
• 38. Youngblood BA, Leung J, Falahati R, et al. Discovery, function, and therapeutic targeting of siglec-8. Cells 2020;10:19. https://doi.org/10.3390/cells10010019
• 39. Stefanski AL, Renecle MD, Kramer A, et al. Siglec-6 signaling uses src kinase tyrosine phosphorylation and SHP-2 recruitment. Cells 2022;11:3427. https://doi.org/10.3390/cells11213427
• 40. Rumer KK, Uyenishi J, Hoffman MC, Fisher BM, Winn VD. Siglec-6 expression is increased in placentas from pregnancies complicated by preterm preeclampsia. Reprod Sci 2013;20:646-653. https://doi.org/10.1177/1933719112461185
• 41. Brinkman Van der Linden ECM, Hurtado Ziola N, Hayakawa T, et al. Human-specific expression of Siglec-6 in the placenta. Glycobiology 2007;17:922-931. https://doi.org/10.1093/glycob/cwm065
• 42. Choi H, Ho M, Adeniji OS, et al. Development of Siglec-9 blocking antibody to enhance anti-tumor immunity. Front Oncol 2021;11:778989. https://doi.org/10.3389/fonc.2021.778989
• 43. Wu Y, Huang W, Xie Y, et al. Siglec-9, a putative immune checkpoint marker for cancer progression across multiple cancer types. Front Mol Biosci 2022;9:743515. https://doi.org/10.3389/fmolb.2022.743515
• 44. Choi HJ, Chung TW, Choi HJ, et al. Increased α2-6 sialylation of endometrial cells contributes to the development of endometriosis. Exp Mol Med 2018;50:1-12. https://doi.org/10.1038/s12276-018-0167-1