[en] MicroRNAs (miRNAs)—a class of small endogenous non-coding RNAs—are widely involved in post-transcriptional gene regulation of numerous physiological processes. High-throughput sequencing revealed that the miR-192 expression level appeared to be significantly higher in the blood exosomes of sows at early gestation than that in non-pregnant sows. Furthermore, miR-192 was hypothesized to have a regulatory role in embryo implantation; however, the target genes involved in exerting the regulatory function of miR-192 required further elucidation.
Methods: In the present study, potential target genes of miR-192 in porcine endometrial epithelial cells (PEECs) were identified through biotin-labeled miRNA pull-down; functional and pathway enrichment analysis was performed via gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment. Bioinformatic analyses were concurrently used to predict the potential target genes associated with sow embryo implantation. In addition, double luciferase reporter vectors, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), and Western blot were performed to verify the targeting and regulatory roles of the abovementioned target genes.
Results: A total of 1688 differentially expressed mRNAs were identified via miRNA pull-down. Through RT-qPCR, the accuracy of the sequencing data was verified. In the bioinformatics analysis, potential target genes of miR-192 appeared to form a dense inter-regulatory network and regulated multiple signaling pathways, such as metabolic pathways and the PI3K-Akt, MAPKs, and mTOR signaling pathways, that are relevant to the mammalian embryo implantation process. In addition, CSK (C-terminal Src kinase) and YY1 (Yin-Yang-1) were predicted to be potential candidates, and we validated that miR-192 directly targets and suppresses the expression of the CSK and YY1 genes.
Conclusion: We screened 1688 potential target genes of miR-192 were screened, and CSK and YY1 were identified as miR-192 target genes. The outcomes of the present study provide novel insights into the regulatory mechanism of porcine embryo implantation and the identification of miRNA target genes.
Precision for document type :
Review article
Disciplines :
Genetics & genetic processes
Author, co-author :
Li, Qingchun; Shihezi University
Gao, Ruonan; Shihezi University
Chen, Yansen ; Université de Liège - ULiège > TERRA Research Centre
Xie, Su; Huazhong Agricultural University: Huazhong Agriculture University
Avila-Bonilla RG, Salas-Benito JS (2022) Interactions of host miRNAs in the flavivirus 3′ UTR genome: from bioinformatics predictions to practical approaches. Front Cell Infect Microbiol 12:976843. 10.3389/fcimb.2022.976843 DOI: 10.3389/fcimb.2022.976843
Lewis BP, Burge CB, Bartel DP (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120(1):15–20. 10.1016/j.cell.2004.12.035 DOI: 10.1016/j.cell.2004.12.035
Lal A, Thomas MP, Altschuler G, Navarro F, O’Day E, Li XL, Concepcion C, Han YC, Thiery J, Rajani DK, Deutsch A, Hofmann O, Ventura A, Hide W, Lieberman J (2011) Capture of microRNA-bound mRNAs identifies the tumor suppressor miR-34a as a regulator of growth factor signaling. PLoS Genet 7(11):e1002363. 10.1371/journal.pgen.1002363 DOI: 10.1371/journal.pgen.1002363
Ørom UA, Nielsen FC, Lund AH (2008) MicroRNA-10a binds the 5′ UTR of ribosomal protein mRNAs and enhances their translation. Mol Cell 30(4):460–471. 10.1016/j.molcel.2008.05.001 DOI: 10.1016/j.molcel.2008.05.001
Schnall-Levin M, Rissland OS, Johnston WK, Perrimon N, Bartel DP, Berger B (2011) Unusually effective microRNA targeting within repeat-rich coding regions of mammalian mRNAs. Genome Res 21(9):1395–1403. 10.1101/gr.121210.111 DOI: 10.1101/gr.121210.111
Ito Y, Inoue A, Seers T, Hato Y, Igarashi A, Toyama T, Taganov KD, Boldin MP, Asahara H (2017) Identification of targets of tumor suppressor microRNA-34a using a reporter library system. Proc Natl Acad Sci USA 114(15):3927–3932. 10.1073/pnas.1620019114 DOI: 10.1073/pnas.1620019114
Easow G, Teleman AA, Cohen SM (2007) Isolation of microRNA targets by miRNP immunopurification. RNA (New York) 13(8):1198–1204. 10.1261/rna.563707 DOI: 10.1261/rna.563707
Thomson DW, Bracken CP, Goodall GJ (2011) Experimental strategies for microRNA target identification. Nucleic Acids Res 39(16):6845–6853. 10.1093/nar/gkr330 DOI: 10.1093/nar/gkr330
Awan HM, Shah A, Rashid F, Wei S, Chen L, Shan G (2018) Comparing two approaches of miR-34a target identification, biotinylated-miRNA pulldown vs miRNA overexpression. RNA Biol 15(1):55–61. 10.1080/15476286.2017.1391441 DOI: 10.1080/15476286.2017.1391441
Orom UA, Lund AH (2007) Isolation of microRNA targets using biotinylated synthetic microRNAs. Methods (San Diego) 43(2):162–165. 10.1016/j.ymeth.2007.04.007 DOI: 10.1016/j.ymeth.2007.04.007
Ren FJ, Yao Y, Cai XY, Fang GY (2021) Emerging role of MiR-192–5p in human diseases. Front Pharmacol 12:614068. 10.3389/fphar.2021.614068 DOI: 10.3389/fphar.2021.614068
Bian Q, Chen B, Weng B, Chu D, Tang X, Yan S, Yin Y, Ran M (2021) circBTBD7 promotes immature porcine sertoli cell growth through modulating miR-24-3p/MAPK7 axis to inactivate p38 MAPK signaling pathway. Int J Mol Sci 22(17):9385. 10.3390/ijms22179385 DOI: 10.3390/ijms22179385
Wang B, Xu S, Wang T, Xu K, Yin L, Li X, Sun R, Pu Y, Zhang J (2022) LincRNA-p21 promotes p21-mediated cell cycle arrest in benzene-induced hematotoxicity by sponging miRNA-17-5p. Environ Pollut (Barking) 296:118706. 10.1016/j.envpol.2021.118706 DOI: 10.1016/j.envpol.2021.118706
Phatak P, Donahue JM (2017) Biotinylated micro-RNA pull down assay for identifying miRNA targets. Bio-protocol 7(9):e2253. 10.21769/BioProtoc.2253 DOI: 10.21769/BioProtoc.2253
Zhang Y, Zhang D, Xu Y, Qin Y, Gu M, Cai W, Bai Z, Zhang X, Chen R, Sun Y, Wu Y, Wang Z (2022) Selection of cashmere fineness functional genes by translatomics. Front Genet 12:775499. 10.3389/fgene.2021.775499 DOI: 10.3389/fgene.2021.775499
Zhang Y, Duan X, Cao R, Liu HL, Cui XS, Kim NH, Rui R, Sun SC (2014) Small GTPase RhoA regulates cytoskeleton dynamics during porcine oocyte maturation and early embryo development. Cell Cycle (Georgetown) 13(21):3390–3403. 10.4161/15384101.2014.952967 DOI: 10.4161/15384101.2014.952967
Jalali BM, Likszo P, Andronowska A, Skarzynski DJ (2018) Alterations in the distribution of actin and its binding proteins in the porcine endometrium during early pregnancy: possible role in epithelial remodeling and embryo adhesion. Theriogenology 116:17–27. 10.1016/j.theriogenology.2018.05.004 DOI: 10.1016/j.theriogenology.2018.05.004
Han LW, Shi Y, Paquette A, Wang L, Bammler TK, Mao Q (2021) Key hepatic metabolic pathways are altered in germ-free mice during pregnancy. PLoS ONE 16(3):e0248351. 10.1371/journal.pone.0248351 DOI: 10.1371/journal.pone.0248351
Yamamoto Y, Maruyama T, Sakai N, Sakurai R, Shimizu A, Hamatani T, Masuda H, Uchida H, Sabe H, Yoshimura Y (2002) Expression and subcellular distribution of the active form of c-Src tyrosine kinase in differentiating human endometrial stromal cells. Mol Hum Reprod 8(12):1117–1124. 10.1093/molehr/8.12.1117 DOI: 10.1093/molehr/8.12.1117
Nagashima T, Maruyama T, Uchida H, Kajitani T, Arase T, Ono M, Oda H, Kagami M, Masuda H, Nishikawa S, Asada H, Yoshimura Y (2008) Activation of SRC kinase and phosphorylation of signal transducer and activator of transcription-5 are required for decidual transformation of human endometrial stromal cells. Endocrinology 149(3):1227–1234. 10.1210/en.2007-1217 DOI: 10.1210/en.2007-1217
Wang Y, Yang D, Zhu R, Dai F, Yuan M, Zhang L, Zheng Y, Liu S, Yang X, Cheng Y (2022) YY1/ITGA3 pathway may affect trophoblastic cells migration and invasion ability. J Reprod Immunol 153:103666. 10.1016/j.jri.2022.103666 DOI: 10.1016/j.jri.2022.103666
Yang D, Ding J, Wang Y, Yuan M, Xian S, Zhang L, Liu S, Dai F, Wang F, Zheng Y, Zhao X, Liao S, Cheng Y (2020) YY1-PVT1 affects trophoblast invasion and adhesion by regulating mTOR pathway-mediated autophagy. J Cell Physiol 235(10):6637–6646. 10.1002/jcp.29560 DOI: 10.1002/jcp.29560
Li R, Song XT, Guo SW, Zhao N, He M, He CQ, Ding NZ (2021) YY1 and RTCB in mouse uterine decidualization and embryo implantation. Reproduction (Cambridge) 162(6):461–472. 10.1530/REP-21-0281 DOI: 10.1530/REP-21-0281
Akbalik ME, Ketani MA (2013) Expression of epidermal growth factor receptors and epidermal growth factor, amphiregulin and neuregulin in bovine uteroplacental tissues during gestation. Placenta 34(12):1232–1242. 10.1016/j.placenta.2013.09.019 DOI: 10.1016/j.placenta.2013.09.019
Monsivais D, Clementi C, Peng J, Fullerton PT Jr, Prunskaite-Hyyryläinen R, Vainio SJ, Matzuk MM (2017) BMP7 induces uterine receptivity and blastocyst attachment. Endocrinology 158(4):979–992. 10.1210/en.2016-1629 DOI: 10.1210/en.2016-1629
Khanbarari F, Ghasemi N, Vakili M, Samadi M (2021) Association of the single nucleotide polymorphism C1858T of the PTPN22 gene with unexplained recurrent pregnancy loss: a case-control study. Int J Reprod Biomed 19(10):873–880. 10.18502/ijrm.v19i10.9819 DOI: 10.18502/ijrm.v19i10.9819
Hua R, Zhang X, Li W, Lian W, Liu Q, Gao D, Wang Y, Lei M (2020) Ssc-miR-21-5p regulates endometrial epithelial cell proliferation, apoptosis and migration via the PDCD4/AKT pathway. J Cell Sci 133(23):248898. 10.1242/jcs.248898 DOI: 10.1242/jcs.248898
Gonzalez TL, Eisman LE, Joshi NV, Flowers AE, Wu D, Wang Y, Santiskulvong C, Tang J, Buttle RA, Sauro E, Clark EL, DiPentino R, Jefferies CA, Chan JL, Lin Y, Zhu Y, Afshar Y, Tseng HR, Taylor K, Williams J et al (2021) High-throughput miRNA sequencing of the human placenta: expression throughout gestation. Epigenomics 13(13):995–1012. 10.2217/epi-2021-0055 DOI: 10.2217/epi-2021-0055
Adur MK, Hale BJ, Ross JW (2017) Detection of miRNA in mammalian oocytes and embryos. Methods Mol Biol (Clifton) 1605:63–81. 10.1007/978-1-4939-6988-3_5 DOI: 10.1007/978-1-4939-6988-3_5
Kropp J, Khatib H (2015) Characterization of microRNA in bovine in vitro culture media associated with embryo quality and development. J Dairy Sci 98(9):6552–6563. 10.3168/jds.2015-9510 DOI: 10.3168/jds.2015-9510
Ji D, Jiang L, Li Y (2018) MiR-192-5p suppresses the growth of bladder cancer cells via targeting Yin Yang 1. Hum Cell 31(3):210–219. 10.1007/s13577-018-0201-6 DOI: 10.1007/s13577-018-0201-6
Edwards SM, Cunningham SA, Dunlop AL, Corwin EJ (2017) The maternal gut microbiome during pregnancy. Am J Matern Child Nurs 42(6):310–317. 10.1097/NMC.0000000000000372 DOI: 10.1097/NMC.0000000000000372
Johnson GA, Bazer FW, Seo H (2021) The early stages of implantation and placentation in the pig. Adv Anat Embryol Cell Biol 234:61–89. 10.1007/978-3-030-77360-1_5 DOI: 10.1007/978-3-030-77360-1_5
Yang Y, Wang L, Chen C, Qi H, Baker PN, Liu X, Zhang H, Han TL (2020) Metabolic changes of maternal uterine fluid, uterus, and plasma during the peri-implantation period of early pregnancy in mice. Reprod Sci (Thousand Oaks) 27(2):488–502. 10.1007/s43032-019-00040-5 DOI: 10.1007/s43032-019-00040-5
Xu Y, Sui L, Qiu B, Yin X, Liu J, Zhang X (2019) ANXA4 promotes trophoblast invasion via the PI3K/Akt/eNOS pathway in preeclampsia. Am J Physiol Cell Physiol 316(4):C481–C491. 10.1152/ajpcell.00404.2018 DOI: 10.1152/ajpcell.00404.2018
Gupta K, Sirohi VK, Kumari S, Shukla V, Manohar M, Popli P, Dwivedi A (2018) Sorcin is involved during embryo implantation via activating VEGF/PI3K/Akt pathway in mice. J Mol Endocrinol 60(2):119–132. 10.1530/JME-17-0153 DOI: 10.1530/JME-17-0153
Lanekoff I, Cha J, Kyle JE, Dey SK, Laskin J, Burnum-Johnson KE (2016) Trp53 deficient mice predisposed to preterm birth display region-specific lipid alterations at the embryo implantation site. Sci Rep 6:33023. 10.1038/srep33023 DOI: 10.1038/srep33023
Zeng X, Mao X, Huang Z, Wang F, Wu G, Qiao S (2013) Arginine enhances embryo implantation in rats through PI3K/PKB/mTOR/NO signaling pathway during early pregnancy. Reproduction (Cambridge) 145(1):1–7. 10.1530/REP-12-0254 DOI: 10.1530/REP-12-0254
Lu CW, Yabuuchi A, Chen L, Viswanathan S, Kim K, Daley GQ (2008) Ras-MAPK signaling promotes trophectoderm formation from embryonic stem cells and mouse embryos. Nat Genet 40(7):921–926. 10.1038/ng.173 DOI: 10.1038/ng.173
Zhang JY, Jiang Y, Lin T, Kang JW, Lee JE, Jin DI (2015) Lysophosphatidic acid improves porcine oocyte maturation and embryo development in vitro. Mol Reprod Dev 82(1):66–77. 10.1002/mrd.22447 DOI: 10.1002/mrd.22447
Johnson GA, Burghardt RC, Bazer FW (2014) Osteopontin: a leading candidate adhesion molecule for implantation in pigs and sheep. J Anim Sci Biotechnol 5(1):56. 10.1186/2049-1891-5-56 DOI: 10.1186/2049-1891-5-56
Wang CX, Chen F, Zhang WF, Zhang SH, Shi K, Song HQ, Wang YJ, Kim SW, Guan WT (2018) Leucine promotes the growth of fetal pigs by increasing protein synthesis through the mTOR signaling pathway in longissimus dorsi muscle at late gestation. J Agric Food Chem 66(15):3840–3849. 10.1021/acs.jafc.8b00330 DOI: 10.1021/acs.jafc.8b00330
Kiewisz J, Kaczmarek MM, Andronowska A, Blitek A, Ziecik AJ (2011) Gene expression of WNTs, β-catenin and E-cadherin during the periimplantation period of pregnancy in pigs—involvement of steroid hormones. Theriogenology 76(4):687–699. 10.1016/j.theriogenology.2011.03.022 DOI: 10.1016/j.theriogenology.2011.03.022
Jalali BM, Lukasik K, Witek K, Baclawska A, Skarzynski DJ (2020) Changes in the expression and distribution of junction and polarity proteins in the porcine endometrium during early pregnancy period. Theriogenology 142:196–206. 10.1016/j.theriogenology.2019.09.041 DOI: 10.1016/j.theriogenology.2019.09.041
Kwon SG, Hwang JH, Park DH, Kim TW, Kang DG, Kang KH, Kim IS, Park HC, Na CS, Ha J, Kim CW (2016) Identification of differentially expressed genes associated with litter size in berkshire pig placenta. PLoS ONE 11(4):e0153311. 10.1371/journal.pone.0153311 DOI: 10.1371/journal.pone.0153311
Zheng L, Chen Y, Ye L, Jiao W, Song H, Mei H, Li D, Yang F, Li H, Huang K, Tong Q (2017) miRNA-584-3p inhibits gastric cancer progression by repressing Yin Yang 1-facilitated MMP-14 expression. Sci Rep 7(1):8967. 10.1038/s41598-017-09271-5 DOI: 10.1038/s41598-017-09271-5
Tian FJ, Cheng YX, Li XC, Wang F, Qin CM, Ma XL, Yang J, Lin Y (2016) The YY1/MMP2 axis promotes trophoblast invasion at the maternal-fetal interface. J Pathol 239(1):36–47. 10.1002/path.4694 DOI: 10.1002/path.4694
Liu LP, Gong YB (2018) LncRNA-TCL6 promotes early abortion and inhibits placenta implantation via the EGFR pathway. Eur Rev Med Pharmacol Sci 22(21):7105–7112. 10.26355/eurrev_201811_16242 DOI: 10.26355/eurrev_201811_16242
Donohoe ME, Zhang X, McGinnis L, Biggers J, Li E, Shi Y (1999) Targeted disruption of mouse Yin Yang 1 transcription factor results in peri-implantation lethality. Mol Cell Biol 19(10):7237–7244. 10.1128/MCB.19.10.7237 DOI: 10.1128/MCB.19.10.7237
Roskoski R (2004) Src protein-tyrosine kinase structure and regulation. Biochem Biophys Res Commun 324(4):1155–1164. 10.1016/j.bbrc.2004.09.171 DOI: 10.1016/j.bbrc.2004.09.171
