Animal Bioscience

Asian Australasian Association of Animal Production Societies (아세아태평양축산학회)
권호 표지
DOI QR코드

DOI QR Code

Serum exosomal miR-192 serves as a potential detective biomarker for early pregnancy screening in sows

  • Ruonan Gao (College of Animal Science and Technology, Shihezi University) ;
  • Qingchun Li (College of Animal Science and Technology, Shihezi University) ;
  • Meiyu Qiu (College of Animal Science and Technology, Shihezi University) ;
  • Su Xie (College of Animal Science and Technology, Shihezi University) ;
  • Xiaomei Sun (College of Animal Science and Technology, Shihezi University) ;
  • Tao Huang (College of Animal Science and Technology, Shihezi University)
  • Received : 2022.11.04
  • Accepted : 2023.04.19
  • Published : 2023.09.01
Download PDF
⟨ Previous Next ⟩

Abstract

Objective: The study was conducted to screen differentially expressed miRNAs in sows at early pregnancy by high-throughput sequencing and explore its mechanism of action on embryo implantation. Methods: The blood serum of pregnant and non-pregnant Landrace×Yorkshire sows were collected 14 days after artificial insemination, and exosomal miRNAs were purified for high throughput miRNA sequencing. The expression patterns of 10 differentially expressed (DE) miRNAs were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The qRT-PCR quantified the abundance of serum exosomal miR-192 in pregnant and control sows, and the diagnostic power was assessed by receiver operating characteristic (ROC) analysis. The target genes of DE miRNAs were predicted with bioinformatics software, and the functional and pathway enrichment analysis was performed on gene ontology and the Kyoto encyclopedia of genes and genomes terms. Furthermore, a luciferase reporter system was used to identify the target relation between miR-192 and integrin alpha 4 (ITGA4), a gene influencing embryo implantation in pigs. Finally, the expression levels of miRNAs and the target gene ITGA4 were analyzed by qRT-PCR, and western blot, with the proliferation of BeWo cells detected by cell counting kit-8 (CCK-8). Results: A total of 221 known miRNAs were detected in the libraries of the pregnant and non-pregnant sows, of which 55 were up-regulated and 67 were down-regulated in the pregnant individuals compared with the non-pregnant controls. From these, the expression patterns of 10 DE miRNAs were validated. The qRT-PCR analysis further confirmed a significantly higher expression of miR-192 in the serum exosomes extracted from pregnant sows, when compared to controls. The ROC analysis revealed that miR-192 provided excellent diagnostic accuracy for pregnancy (area under the ROC curve [AUC]=0.843; p>0.001). The dual-luciferase reporter assay indicated that miR-192 directly targeted ITGA4. The protein expression of ITGA4 was reduced in cells that overexpressed miR-192. Overexpression of miR-192 resulted in the decreased proliferation of BeWo cells and regulated the expression of cell cycle-related genes. Conclusion: Serum exosomal miR-192 could serve as a potential biomarker for early pregnancy in pigs. miR-192 targeted ITGA4 gene directly, and miR-192 can regulate cellular proliferation.

Keywords

Acknowledgement

We thank all contributors of the present study. The authors would like to express their gratitude to EditSprings (https://www.editsprings.com/) for the expert linguistic services provided.

References

  1. Van der Pol E, Boing AN, Harrison P, et al. Classification, functions, and clinical relevance of extracellular vesicles. Pharmacol Rev 2012;64:676-705. https://doi.org/10.1124/pr.112.005983 
  2. Johnstone RM, Adam M, Hammond JR, Orr L, Turbide C. Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes). J Biol Chem 1987;262:9412-20. https://doi.org/10.1016/S0021-9258(18)48095-7 
  3. Dhondt B, Van Deun J, Vermaerke S, et al. Urinary extracellular vesicle biomarkers in urological cancers: From discovery towards clinical implementation. Int J Biochem Cell Biol 2018;99:236-56. https://doi.org/10.1016/j.biocel.2018.04.009 
  4. Zhao G, Chao Y, Jing Y, et al. Placental exosome-mediated Bta-miR-499-Lin28B/let-7 axis regulates inflammatory bias during early pregnancy. Cell Death Dis 2018;9:704. https://doi.org/10.1038/s41419-018-0713-8 
  5. Vilella F, Moreno-Moya JM, Balaguer N, et al. Hsa-miR-30d, secreted by the human endometrium, is taken up by the pre-implantation embryo and might modify its transcriptome. Development (Cambridge, England) 2015;142:3210-21. https://doi.org/10.1242/dev.124289 
  6. Smolinska N, Kiezun M, Dobrzyn K, Szeszko K, Maleszka A, Kaminski T. Expression of the orexin system in the porcine uterus, conceptus and trophoblast during early pregnancy. Animal 2015;9:1820-31. https://doi.org/10.1017/S1751731115001020 
  7. Carella AM, Marinelli T, Melfi A, et al. Circulating MicroRNAs as cancer biomarkers: Can They Play a Role in Clinical Practice Short Review. Arch Prev Med 2016;1:004-007.  https://doi.org/10.17352/apm.000002
  8. Stoick-Cooper CL, Weidinger G, Riehle KJ, et al. Distinct Wnt signaling pathways have opposing roles in appendage regeneration. Development 2007;134:479-89. https://doi.org/10.1242/dev.001123 
  9. Inyawilert W, Fu TY, Lin CT, Tang PC. Let-7-mediated suppression of mucin 1 expression in the mouse uterus during embryo implantation. J Reprod Dev 2015;61:138-44. https://doi.org/10.1262/jrd.2014-106 
  10. Kottawatta KSA, So KH, Kodithuwakku SP, Ng EHY, Yeung WSB, Lee KF. MicroRNA-212 regulates the expression of olfactomedin 1 and C-terminal binding protein 1 in human endometrial epithelial cells to enhance spheroid attachment in vitro. Biol Reprod 2015;93:109. https://doi.org/10.1095/biolreprod.115.131334 
  11. Zhang Q. Function and mechanism study of non-coding RNAs miR-148a-3p and lincARDD1 in decidualization of recurrent implantation failure [PhD thesis]. Shandong, China: Shandong University; 2018. 
  12. Ast V, Kordass T, Oswald M, et al. miR-192, miR-200c and miR-17 are fibroblast-mediated inhibitors of colorectal cancer invasion. Oncotarget 2018;9:35559-80. https://doi.org/10.18632/oncotarget.26263 
  13. Shang G, Mi Y, Mei Y, et al. MicroRNA-192 inhibits the proliferation, migration and invasion of osteosarcoma cells and promotes apoptosis by targeting matrix metalloproteinase-11. Oncol Lett 2018;15:7265-72. https://doi.org/10.3892/ol.2018.8239 
  14. Li J, Smyth P, Flavin R, et al. Comparison of mirna expression patterns using total Rna Extracted from matched samples of formalin-fixed paraffin-embedded (FFPE) cells and snap frozen cells. BMC Biotechnol 2007;7:36. https://doi.org/10.1186/1472-6750-7-36 
  15. Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 2009;10:R25. https://doi.org/10.1186/gb-2009-10-3-r25 
  16. Friedlander MR, Mackowiak SD, Li N, et al. Mirdeep2 accurately identifies known and hundreds of novel microrna genes in seven animal clades. Nucleic Acids Res 2012;40:37-52. https://doi.org/10.1093/nar/gkr688 
  17. Yang CX, Du ZQ, Wright EC, Rothschild MF, Prather RS, Ross JW. Small RNA profile of the cumulus-oocyte complex and early embryos in the pig. Biol Reprod 2012;87:117. https://doi.org/10.1095/biolreprod.111.096669 
  18. Wen M, Shen Y, Shi S, Tang T. Mirevo: an integrative microrna evolutionary analysis platform for next-generation sequencing experiments. BMC Bioinformatics 2012;13:140. https://doi.org/10.1186/1471-2105-13-140 
  19. Calabrese JM, Seila AC, Yeo GW, Sharp PA. RNA sequence analysis defines Dicer's role in mouse embryonic stem cells. Proc Natl Acad Sci USA 2007;104:18097-102. https://doi.org/10.1073/pnas.0709193104 
  20. Kanehisa M, Araki M, Goto S, et al. KEGG for linking genomes to life and the environment. Nucleic Acids Res 2008;36(Suppl):D480-4. https://doi.org/10.1093/nar/gkm882
  21. Wu J, Mao X, Cai T, Luo J, Wei L. KOBAS server: a web-based platform for automated annotation and pathway identification. Nucleic Acids Res 2006;34(Suppl 2):W720-4. https://doi.org/10.1093/nar/gkl167 
  22. Enright AJ, John B, Gaul U, Tuschl T, Sander C, Marks DS. MicroRNA targets in Drosophila. Genome Biol 2003;5:R1. https://doi.org/10.1186/gb-2003-5-1-r1 
  23. Kruger J, Rehmsmeier M. RNAhybrid: microRNA target prediction easy, fast and flexible. Nucleic Acids Res 2006; 34(suppl_2):W451-4. https://doi.org/10.1093/nar/gkl243 
  24. Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB. Prediction of mammalian microRNA targets. Cell 2003;115:787-98. https://doi.org/10.1016/s0092-8674(03)01018-3 
  25. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 2001;25:402-8. https://doi.org/10.1006/meth.2001.1262 
  26. Cunningham NF, Hattersley JJ, Wrathall AE. Pregnancy diagnosis in sows based on serum oestrone sulphate concentration. Vet Rec 1983;113:229-33. https://doi.org/10.1136/vr.113.11.229 
  27. Moriyoshi M, Nozoki K, Ohtaki T, et al. Measurement of gestagen concentration in feces using a bovine milk progesterone quantitative test eia kit and its application to early pregnancy diagnosis in the sow. J Vet Med Sci 1997;59:695-701. https://doi.org/10.1292/jvms.59.695 
  28. Szenci O, Palme R, Taverne MAM, Varga J, Meersma N, Wissink E. Evaluation of false ultrasonographic pregnancy diagnoses in sows by measuring the concentration of unconjugated estrogens in feces. Theriogenology 1997;48:873-82. https://doi.org/10.1016/s0093-691x(97)00308-7 
  29. Choi H, Kiesenhofer E, Gantner H, et al. Pregnancy diagnosis in sows by estimation of oestrogens in blood, urine or faeces. Anim Reprod Sci 1987;15:209-16.  https://doi.org/10.1016/0378-4320(87)90043-1
  30. Williams SI, Pieyro P, Sota RLDL. Accuracy of pregnancy diagnosis in swine by ultrasonography. Can Vet J 2008;49:269-73. 
  31. Gallo A, Tandon M, Alevizos I, et al. The majority of micrornas detectable in serum and saliva is concentrated in exosomes. Plos One 2012;7:e30679. https://doi.org/10.1371/journal.pone.0030679 
  32. Li JY, Yong TY, Michael MZ, et al. Micrornas: Are they the missing link between hypoxia and pre-eclampsia. Hypertens Pregnancy 2014;33:102-14. https://doi.org/10.3109/10641955.2013.832772 
  33. Mayor-lynn K, Toloubeydokhti T, Cruz AC, Chegini N. Expression profile of micrornas and mrnas in human placentas from pregnancies complicated by preeclampsia and preterm labor. Reprod Sci 2011;18:46-56. https://doi.org/10.1177/1933719110374115 
  34. Zhao Z, Moley KH, Gronowski AM. Diagnostic potential for mirnas as biomarkers for pregnancy-specific diseases. Clin Biochem 2013;46:953-60. https://doi.org/10.1016/j.clinbiochem.2013.01.026 
  35. Fu G, Ye G, Nadeem L, et al. MicroRNA-376c impairs transforming growth factor-β and nodal signaling to promote trophoblast cell proliferation and invasion. Hypertension 2013; 61:864-72. https://doi.org/10.1161/HYPERTENSIONAHA.111.203489 
  36. Su L, Zhao S, Zhu M, Yu M. Differential expression of micrornas in porcine placentas on days 30 and 90 of gestation. Reprod Fertil Dev 2010;22:1175-82. https://doi.org/10.1071/RD10046 
  37. Ioannidis J, Donadeu FX. Circulating miRNA signatures of early pregnancy in cattle. BMC Genomics 2016;17:184. https://doi.org/10.1186/s12864-016-2529-1 
  38. Fiandanese N, Viglino A, Strozzi F. Circulating microRNAs as potential biomarkers of early pregnancy in high producing dairy cows. Reprod Fertil Dev 2015;28:165. https://doi.org/10.1071/RDv28n2Ab71
  39. Lim HJ, Kim HJ, Lee JH, et al. Identification of plasma miRNA biomarkers for pregnancy detection in dairy cattle. J Anim Reprod Biotechnol 2021;36:35-44. https://doi.org/10.12750/JARB.36.1.35 
  40. Klohonatz KM, Cameron AD, Hergenreder JR, et al. Circulating miRNAs as potential alternative cell signaling associated with maternal recognition of pregnancy in the mare. Biol Reprod 2016;95:124. https://doi.org/10.1095/biolreprod.116.142935 
  41. Huang Q, Hou S, Zhu X, Liu S. MicroRNA-192 promotes the development of nasopharyngeal carcinoma through targeting RB1 and activating PI3K/AKT pathway. World J Surg Oncol 2020;18:29. https://doi.org/10.1186/s12957-020-1798-y 
  42. Cleys ER, Halleran JL, Mcwhorter E, et al. Identification of micrornas in exosomes isolated from serum and umbilical cord blood, as well as placentomes of gestational day 90 pregnant sheep. Mol Reprod Dev 2014;81:983-93. https://doi.org/10.1002/mrd.22420 
  43. Chung AC, Huang XR, Meng X, Lan HY. miR-192 mediates TGF-β/smad3-driven renal fibrosis. J Am Soc Nephrol 2010;21:1317-25. https://doi.org/10.1681/ASN.2010020134 
  44. He B, Chang Y, Yang C, et al. Adenylate cyclase 7 regulated by miR-192 promotes ATRA-induced differentiation of acute promyelocytic leukemia cells. Biochem Biophys Res Commun 2018;506:543-7. https://doi.org/10.1016/j.bbrc.2018.10.125 
  45. Jia B, Liu Y, Li Q, et al. Altered miRNA and mRNA expression in sika deer skeletal muscle with age. Genes 2020;11:172. https://doi.org/10.3390/genes11020172 
  46. Liang J, Cao D, Zhang X, et al. miR-192-5p suppresses uterine receptivity formation through impeding epithelial transformation during embryo implantation. Theriogenology 2020;157;360-71. https://doi.org/10.1016/j.theriogenology.2020.08.009 
  47. Ma F, Li T, Zhang H, Wu G. MiR-30s family inhibit the proliferation and apoptosis in human coronary artery endothelial cells through targeting the 3' UTR region of ITGA4 and PLCG1. J Cardiovasc Pharmacol 2016;68:327-33. https://doi.org/10.1097/FJC.0000000000000419 
  48. Merviel P, Challier JC, Carbillon L, Foidart JM, Uzan S. The role of integrins in human embryo implantation. Fetal Diagn Ther 2001;16:364-71. https://doi.org/10.1159/000053942 
  49. Basak S, Dhar R, Das C. Steroids modulate the expression of α4 integrin in mouse blastocysts and uterus during implantation. Biol Reprod 2002;66:1784-9. https://doi.org/10.1095/biolreprod66.6.1784 
  50. Song F, Li Y, Wang GM, et al. Effects of integrins αv and α4 in mouse blastocyst implantation. Chinese J Anat 2003;322-6. 
  51. Chen ZJ, Yan YJ, Shen H, et al. miR-192 is overexpressed and promotes cell proliferation in prostate cancer. Med Princ Pract 2019;28:124-32. https://doi.org/10.1159/000496206 
  52. Mao Q, Chen C, Liang H, Zhong S, Cheng X, Li L. Astragaloside IV inhibits excessive mesangial cell proliferation and renal fibrosis caused by diabetic nephropathy via modulation of the TGF β1/Smad/miR 192 signaling pathway. Exp Ther Med 2019;18:3053-61. https://doi.org/10.3892/etm.2019.7887 
  53. Garcia-Riart B, Lorda-Diez CI, Marin-Llera JC, Garcia-Porrero JA, Hurle JM, Montero JA. Interdigital tissue remodelling in the embryonic limb involves dynamic regulation of the miRNA profiles. J Anat 2017;231:275-86. https://doi.org/10.1111/joa.12629 
  54. Woo I, Christenson LK, Gunewardena S, et al. Micro-RNAs involved in cellular proliferation have altered expression profiles in granulosa of young women with diminished ovarian reserve. J Assist Reprod Genet 2018;35:1777-86. https://doi.org/10.1007/s10815-018-1239-9