Comprehensive analysis of genes related to endometrial receptivity and alternative splicing events in northwest Tibetan cashmere goats

doi:10.12307/2025.010

Abstract

Abstract: BACKGROUND: Endometrial receptivity is a key factor in embryo implantation in northwest Tibetan cashmere goats, and the expression of genes related to endometrial receptivity and their variable splicing are still unclear.
OBJECTIVE: To analyze and explore genes and variable splicing events related to endometrial receptivity in northwest Tibetan cashmere goats.
METHODS: On days 5 and 15 of pregnancy (representing pre receptive endometrium group and receptive endometrium group), three northwest Tibetan cashmere goats were randomly selected. Endometrial tissue was collected and stained with hematoxylin and eosin to observe tissue morphology. Immunohistochemical staining was used to detect the expression of endometrial receptive marker proteins leukemia inhibitory factor and vascular endothelial growth factor. After the total RNA was extracted and the quality test was qualified, transcriptome sequencing was performed to search differentially expressed mRNAs, lncRNAs, circRNAs, and miRNAs, perform functional prediction, and analyze alternative splicing mRNAs and lncRNAs related to endometrial receptivity.
RESULTS AND CONCLUSION: (1) Compared with the pre receptive endometrium group, the expression levels of leukemia inhibitory factor and vascular endothelial growth factor proteins in the endometrial tissue of the receptive endometrium group were significantly increased. (2) The sequencing results showed that the differentially expressed genes were mostly mRNA and lncRNA genes, including 250 upregulated mRNAs, 193 upregulated lncRNAs, 135 downregulated mRNAs, and 123 downregulated lncRNAs, which were significantly enriched in the Wnt, Hedgehog, and Hippo signaling pathways. (3) Alternative splicing event analysis uncovered 8 differentially expressed variable splicing transcripts, which were all mRNA transcripts, including 2 downregulated and 6 upregulated, and were significantly associated with vascular endothelial growth factor receptor signaling, cell motility, and embryonic development.

Key words: northwest Tibetan cashmere goat, endometrial receptivity, transcriptome sequencing, differentially expressed gene, alternative splicing, leukemia inhibitory factor, vascular endothelial growth factor

CLC Number:

De Ji, Suo Langda, Wei Yuchen, Wang Bin, Awangcuoji, Renqingcuomu, Cui Jiuzeng, Zhang Lei, Ba Gui. Comprehensive analysis of genes related to endometrial receptivity and alternative splicing events in northwest Tibetan cashmere goats[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(7): 1429-1436.

Figures/Tables 4

2.2 差异表达miRNA分子与组织、生物体和系统发育显著相关 RNA高通量测序结果表明，在PE和RE两组之间，只有4个显著上调和4个下调的miRNA，见图2A。功能注释显示，这些miRNA与组织、生物体和系统发育显著相关，并富集于癌症相关通路和激酶通路，包括磷脂酰肌醇3激酶(phosphatidylinositol-3-kinase，PI3K)/蛋白激酶B(protein kinase B，AKT)信号通路、丝裂原活化蛋白激酶(mitogen-activated protein kinase，MAPK)信号通路和Hippo信号通路，见图2B。 2.3 差异表达的circRNA与抗原呈递显著相关，并在免疫排斥相关通路中富集与PE组织相比，RE组织中存在11个显著上调和8个下调的circRNA，见图3A。功能注释表明，这些circRNA与抗原呈递复合物和抗原处理显著相关，并在免疫排斥相关途径中富集，如IgA产生、同种异体排斥和移植物抗宿主病等，见图3B。 2.4 差异表达的mRNA与细胞运动和成形显著相关，并富集于甘油磷脂和嘧啶代谢相关途径 PE和RE组织中有17 604个编码基因的转录本(即mRNA)，其中250个显著上调，135个下调(RE/PE)，见图4A。GO分析表明，它们与细胞投影、基于微管的过程和纤毛显著相关，而纤毛在细胞运动和成形中非常重要；KEGG分析显示，富集于甘油磷脂和嘧啶代谢相关途径，见图4B。 2.5 差异表达的lncRNA富集于Wnt、Hedgehog和Hippo信号通路与PE组织相比，RE组织中分别有193个显著上调和123个显著下调的lncRNA，见图5A。GO分析表明，这些lncRNA与对多种信号的响应以及细胞周期等生物学过程的调节显著相关；KEGG分析表明，它们富集于Wnt、Hedgehog和 Hippo信号通路，见图5B。 2.6 靶基因预测和竞争性内源RNA调控网络分析根据靶基因预测结果，统计lncRNA和mRNA上miRNA反应元件的个数，结果见图6。在总共835个lncRNA中预测到218个含有miRNA反应元件的lncRNA。具有2个miRNA反应元件的lncRNA数量最多，有49个；具有8个miRNA反应元件的lncRNA数量最少，有8个，见图6A。共计165个mRNA中预测到73个含有miRNA反应元件的mRNA。具有3个miRNA反应元件的mRNA有26个；具有6个miRNA反应元件的mRNA有1个，见图6B。符合敏感相关性> 0.3的lncRNA-miRNA-mRNA有15组。一共包含4个lncRNA、6个mRNA和4个共有的miRNA。MSTRG.21700.7分别与ENSCHIG00000021609、ENSCHIG00000011144和ENSCHIG00000007466都存在内源性竞争关系。 2.7 差异可变剪接事件与蛋白代谢和催化活性显著相关可变剪接事件分析表明，外显子跳过类型的可变剪接事件在PE和RE组织的可变剪接事件中居绝对主导地位，见图7A。PE和RE组织之间存在8个差异可变剪接基因，其中2个下调、6个上调，涉及的基因包括TMEM132C、MATN4、 EFEMP1、MAK、ADAM22、LRRC34、RHBDL3和一个未命名基因ENSCHIG00000020109，见图7B。GO分析显示，差异可变剪接基因与蛋白代谢和催化活性显著相关，见图7C。 "

References

[1] WANG W, LI Z, XIE G, et al. Convergent Genomic Signatures of Cashmere Traits: Evidence for Natural and Artificial Selection. Int J Mol Sci. 2023;24(2):1165.
[2] LI C, WU Y, CHEN B, et al. Markhor-derived Introgression of a Genomic Region Encompassing PAPSS2 Confers High-altitude Adaptability in Tibetan Goats. Mol Biol Evol. 2022;39(12):msac253.
[3] ASHARY N, TIWARI A, MODI D. Embryo Implantation: War in Times of Love. Endocrinology. 2018;159(2):1188-1198.
[4] LESSEY BA, YOUNG SL. What exactly is endometrial receptivity? Fertil Steril. 2019;111(4):611-617.
[5] WANG W, ZANG X, LI Y, et al. Integrating Analysis to Identify Differential circRNAs Involved in Goat Endometrial Receptivity. Int J Mol Sci. 2023; 24(2):1531.
[6] LIU H, WANG C, LI Z, et al. Transcriptomic Analysis of STAT1/3 in the Goat Endometrium During Embryo Implantation. Front Vet Sci. 2021; 8:757759.
[7] ZHAO Y, RICHING AS, KNIGHT WE, et al. Cardiomyocyte-Specific Long Noncoding RNA Regulates Alternative Splicing of the Triadin Gene in the Heart. Circulation. 2022;146(9):699-714.
[8] LIU X, ZHANG L, YANG L, et al. miR-34a/c induce caprine endometrial epithelial cell apoptosis by regulating circ-8073/CEP55 via the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways. J Cell Physiol. 2020; 235(12):10051-10067.
[9] CUI J, LIU X, YANG L, et al. MiR-184 Combined with STC2 Promotes Endometrial Epithelial Cell Apoptosis in Dairy Goats via RAS/RAF/MEK/ERK Pathway. Genes (Basel). 2020;11(9):1052.
[10] ZHANG L, LIU X, LIU J, et al. miR-26a promoted endometrial epithelium cells (EECs) proliferation and induced stromal cells (ESCs) apoptosis via the PTEN-PI3K/AKT pathway in dairy goats. J Cell Physiol. 2018; 233(6):4688-4706.
[11] HE M, LI L, WEI X, et al. Xiaoyao powder improves endometrial receptivity via VEGFR-2-mediated angiogenesis through the activation of the JNK and P38 signaling pathways. J Ethnopharmacol. 2022;282: 114580.
[12] LI L, JIANG H, WEI X, et al. Bu Shen Zhu Yun Decoction Improves Endometrial Receptivity via VEGFR-2-Mediated Angiogenesis. Evid Based Complement Alternat Med. 2019;2019:3949824.
[13] LIU J, XIAO Q, XIAO J, et al. Wnt/β-catenin signalling: function, biological mechanisms, and therapeutic opportunities. Signal Transduct Target Ther. 2022;7(1):3.
[14] TEPEKOY F, AKKOYUNLU G, DEMIR R. The role of Wnt signaling members in the uterus and embryo during pre-implantation and implantation. J Assist Reprod Genet. 2015;32(3):337-346.
[15] YI T, LIU M, LI X, et al. Benzo(a)pyrene inhibits endometrial cell apoptosis in early pregnant mice via the WNT5A pathway. J Cell Physiol. 2019;234(7):11119-11129.
[16] HUANG K, CHEN G, FAN W, et al. miR-23a-3p increases endometrial receptivity via CUL3 during embryo implantation. J Mol Endocrinol. 2020;65(2):35-44.
[17] WU Z, GUAN KL. Hippo Signaling in Embryogenesis and Development. Trends Biochem Sci. 2021;46(1):51-63.
[18] FU M, HU Y, LAN T, et al. The Hippo signalling pathway and its implications in human health and diseases. Signal Transduct Target Ther. 2022;7(1):376.
[19] YUE C, CHEN ACH, TIAN S, et al. Human embryonic stem cell-derived blastocyst-like spheroids resemble human trophectoderm during early implantation process. Fertil Steril. 2020;114(3):653-664.e6.
[20] CRITCHLEY HOD, MAYBIN JA, ARMSTRONG GM, et al. Physiology of the Endometrium and Regulation of Menstruation. Physiol Rev. 2020; 100(3):1149-1179.
[21] OGHBAEI F, ZAREZADEH R, JAFARI-GHARABAGHLOU D, et al. Epithelial-mesenchymal transition process during embryo implantation. Cell Tissue Res. 2022;388(1):1-17.
[22] MAZIN PV, KHAITOVICH P, CARDOSO-MOREIRA M, et al. Alternative splicing during mammalian organ development. Nat Genet. 2021;53(6): 925-934.
[23] WRIGHT CJ, SMITH CWJ, JIGGINS CD. Alternative splicing as a source of phenotypic diversity. Nat Rev Genet. 2022;23(11):697-710.
[24] OLTHOF AM, WHITE AK, KANADIA RN. The emerging significance of splicing in vertebrate development. Development. 2022;149(19): dev200373.
[25] SONG H, WANG L, CHEN D, et al. The Function of Pre-mRNA Alternative Splicing in Mammal Spermatogenesis. Int J Biol Sci. 2020;16(1):38-48.
[26] MESEGUER M, PELLICER A, SIMÓN C. MUC1 and endometrial receptivity. Mol Hum Reprod. 1998;4(12):1089-1098.