Roles of miR-3604 on the Receptivity, Proliferation, and Apoptosis of Bovine Endometrial Epithelial Cells

doi:10.13560/j.cnki.biotech.bull.1985.2024-0245

Abstract

Abstract:

【Objective】 To investigate the role of miR-3604 in the receptivity of bovine endometrial epithelial cells（bEECs）.【Method】Progesterone（P4）and interferon tau（IFN-τ）were used to induce bEECs to construct a tolerant cell model. On this basis and after the overexpression and interference of bovine miR-3604 in the tolerant bEECs, the RT-qPCR, flow cytometry, EdU and CCK8 techniques were used to detect the receptivity of bovine miR-3604 in bEECs. Role in cell proliferation and apoptosis. 【Result】The expressions of receptive marker gene HOXA10, IL-6 and VEGF significantly reduced in the miR-3604 overexpression group of receptive bEECs（P < 0.05）. The expressions of apoptosis marker gene CASP3 and BAX were significantly up-regulated（P < 0.05）. The apoptosis rate of acceptive bEECs significantly decreased（P < 0.01）, while the cell proliferation rate significantly increased（P < 0.05）. At the same time, the results of the miR-3604 interference experiment were contrary to the results of the overexpression experiment.【Conclusion】The expression of miR-3604 in receptive bEECs increased, which may inhibit the receptivity and apoptosis of bEECs, and promote the proliferation of bEECs.

Key words: miR-3604, endometrial receptivity, bovine endometrial epithelial cells, cell proliferation, cell apoptosis

JI Zhong-xiang, LUORENG Zhuo-ma, LI Yu-hang, WANG Yu-mei, HU Xi-min, LI Yan-qing, WANG Xing-ping. Roles of miR-3604 on the Receptivity, Proliferation, and Apoptosis of Bovine Endometrial Epithelial Cells[J]. Biotechnology Bulletin, 2024, 40(12): 291-298.

Figures/Tables 7

Table 1 Primer information for RT-qPCR

引物名称Primer name	序列号Serial number	引物序列Primer sequence（5'-3'）	产物长度Product length/bp
miR-3604	NR_036382.1	F：AGTGCAGGGTCCGAGGTATT R：GCCTCATAACCAATGTGCAG	63
HOXA10	NM_001105017.1	F：TTTCCTTGGCAAAGAGAAGGGCTTG R：GACCTTACACAAACTGGAAGAGAC	188
IL-6	NM_173923.2	F：CACTCCATTCGCTGTCT R：GTGTCTCCTTGCTGCTT	227
VEGF	NM_001316955.1	F：GTCTACCAGCGCAGCTTCTG R：TGCTGGCTTTGGTGAGGTT	212
LIF	NM_173931.1	F：GGTCTTGGCGGCAGGAGTT R：GTGGCACAGGTGGCGTTGA	102
CASP3	NM_001077840.1	F：AAGATTTAGTGCCGATGC R：GACCACCAAGTTCTAGGATA	175
BAX	NM_173894.1	F：GCAAACTGGTGCTCAAGG R：GCACTCCAGCCACAAAGA	238
GAPDH	NM_001034034.2	F：GGCATCGTGGAGGGACTTATG R：GCCAGTGAGCTTCCCGTTGAG	186
RPS18	NM_001033614.2	F：GTGGTGTTGAGGAAAGCAGACA R：TGATCACACGTTCCACCTCATC	79

Fig. 1 Expressions of cell receptivity marker genes and miR-3604 in bEECs receptivity models A, B and C: Expressions of receptive marker genes IL-6, VEGF, and LIF. D: Expression of miR-3604, *P <0.05, **P <0.01, the same below

Fig. 2 Overexpression and interference efficiency detection of miR-3604 in bEECs A, B, C and D: Fluorescence observation of bEECs transfected with miR-3604（100×）; E: mimic transfection efficiency; F: inhibitor transfection efficiency

Fig. 3 Effect of miR-3604 on the expression of receptivity marker gene in bEECs A, B and C: The expressions of VEGF, HOXA10, and IL-6 in the miR-3604 overexpression group. D, E and F: The expressions of VEGF, HOXA10, and IL-6 in the miR-3604 inhibitor group

Fig. 4 Effect of miR-3604 on the apoptosis of receptive bEECs A-D: Expressions of CASP3 and BAX genes related to cell apoptosis detected by RT-qPCR. E-J: Cell apoptosis rate detected by flow cytometry

Fig. 5 Effect of miR-3604 on the proliferation of receptive bEECs A: Fluorescence observation of proliferating cells. B and C: Percentage of EdU positive cells

Fig. 6 Effects of miR-3604 on the viability of the receptive bEECs A: Effect of miR-3604 overexpression on the viability of bEECs. B: Effect of miR-3604 interference on the viability of bEECs

References 39

[1]	Kaya A, Dogan S, Vargovic P, et al. Sperm proteins ODF2 and PAWP as markers of fertility in breeding bulls[J]. Cell Tissue Res, 2022, 387(1): 159-171.
[2]	秦雪, 冯瑞, 李琦, 等. 氨离子对奶牛子宫内膜上皮细胞凋亡和容受性的影响[J]. 中国兽医学报, 2022, 42(3): 535-540.
	Qin X, Feng R, Li Q, et al. Effects of ammonia on apoptosis and receptivity of endometrial epithelial cells of dairy cows[J]. Chin J Vet Sci, 2022, 42(3): 535-540.
[3]	Zhang S, Lin HY, Kong SB, et al. Physiological and molecular determinants of embryo implantation[J]. Mol Aspects Med, 2013, 34(5): 939-980. doi: 10.1016/j.mam.2012.12.011 pmid: 23290997
[4]	Lonergan P. Influence of progesterone on oocyte quality and embryo development in cows[J]. Theriogenology, 2011, 76(9): 1594-1601. doi: 10.1016/j.theriogenology.2011.06.012 pmid: 21855985
[5]	余婕, 胡修忠, 向敏, 等. 精氨酸对干扰素-tau处理牛子宫内膜上皮细胞基因表达的影响[J]. 中国畜牧兽医, 2021, 48(7): 2495-2503. doi: 10.16431/j.cnki.1671-7236.2021.07.025
	Yu J, Hu XZ, Xiang M, et al. Effects of arginine on expression of genes in bovine endometrial epithelial cells treated with interferon-tau[J]. China Anim Husb Vet Med, 2021, 48(7): 2495-2503.
[6]	Shekibi M, Heng S, Nie GY. MicroRNAs in the regulation of endometrial receptivity for embryo implantation[J]. Int J Mol Sci, 2022, 23(11): 6210.
[7]	Lyu SJ, Zhai YY, Zhu XT, et al. Bta-miR-200b promotes endometrial epithelial cell apoptosis by targeting MYB in cattle[J]. Theriogenology, 2023, 195: 77-84. doi: 10.1016/j.theriogenology.2022.10.006 pmid: 36332375
[8]	Feng F, Li YX, Wang JP, et al. LncRNA CA12-AS1 targets miR-133a to promote LPS-induced inflammatory response in bovine mammary epithelial cells[J]. Int J Biol Macromol, 2024, 261(Pt 1): 129710.
[9]	Ostovar T, Zadehbagheri S, Hekmatimoghaddam SH. Comparison of different types of liposomal nano structures for microRNA transfection to human mesenchymal stem cell line S1939[J]. Nucleosides Nucleotides Nucleic Acids, 2023, 42(3): 217-233.
[10]	Huang H, Li XY, Wang ZM, et al. Anti-inflammatory effect of selenium on lead-induced testicular inflammation by inhibiting NLRP3 inflammasome activation in chickens[J]. Theriogenology, 2020, 155: 139-149. doi: S0093-691X(20)30365-4 pmid: 32673849
[11]	赵丽, 杨洋, 温传俊. 茎-环RT-PCR法定量miRNA-421的引物设计[J]. 南京师大学报: 自然科学版, 2012, 35(2): 83-88.
	Zhao L, Yang Y, Wen CJ. Stem-loop real-time quantitative PCR for quantification of miRNA-421 by specific primers[J]. J Nanjing Norm Univ Nat Sci Ed, 2012, 35(2): 83-88.
[12]	Tranguch S, Daikoku T, Guo Y, et al. Molecular complexity in establishing uterine receptivity and implantation[J]. Cell Mol Life Sci, 2005, 62(17): 1964-1973. doi: 10.1007/s00018-005-5230-0 pmid: 16143898
[13]	Lonergan P, Sánchez JM. Symposium review: Progesterone effects on early embryo development in cattle[J]. J Dairy Sci, 2020, 103(9): 8698-8707. doi: S0022-0302(20)30510-5 pmid: 32622590
[14]	李琦, 秦雪, 冯瑞, 等. 孕酮对奶牛子宫内膜上皮细胞凋亡和容受性的影响[J]. 中国兽医学报, 2022, 42(9): 1915-1922.
	Li Q, Qin X, Feng R, et al. Effects of progesterone on apoptosis and receptivity of endometrial epithelial cells in dairy cows[J]. Chin J Vet Sci, 2022, 42(9): 1915-1922.
[15]	Garcia-Ispierto I, López-Helguera I, Serrano-Pérez B, et al. Progesterone supplementation during the time of pregnancy recognition after artificial insemination improves conception rates in high-producing dairy cows[J]. Theriogenology, 2016, 85(7): 1343-1347. doi: 10.1016/j.theriogenology.2015.12.021 pmid: 26786244
[16]	Roberts RM. Interferon-tau, a Type 1 interferon involved in maternal recognition of pregnancy[J]. Cytokine Growth Factor Rev, 2007, 18(5/6): 403-408.
[17]	Dorniak P, Bazer FW, Spencer TE. Physiology and Endocrinology Symposium: biological role of interferon tau in endometrial function and conceptus elongation[J]. J Anim Sci, 2013, 91(4): 1627-1638. doi: 10.2527/jas.2012-5845 pmid: 23097402
[18]	Schulte MMB, Tsai JH, Moley KH. Obesity and PCOS: the effect of metabolic derangements on endometrial receptivity at the time of implantation[J]. Reprod Sci, 2015, 22(1): 6-14. doi: 10.1177/1933719114561552 pmid: 25488942
[19]	Diedrich K, Fauser BCJM, Devroey P, et al. The role of the endometrium and embryo in human implantation[J]. Hum Reprod Update, 2007, 13(4): 365-377. doi: 10.1093/humupd/dmm011 pmid: 17548368
[20]	Zhang L, Liu XR, Liu JZ, 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]. J Cell Physiol, 2018, 233(6): 4688-4706. doi: 10.1002/jcp.26252 pmid: 29115668
[21]	Eskandari E, Eaves CJ. Paradoxical roles of caspase-3 in regulating cell survival, proliferation, and tumorigenesis[J]. J Cell Biol, 2022, 221(6): e202201159.
[22]	Feng R, Qin X, Li Q, et al. Progesterone regulates inflammation and receptivity of cells via the NF-κB and LIF/STAT3 pathways[J]. Theriogenology, 2022, 186: 50-59. doi: 10.1016/j.theriogenology.2022.04.005 pmid: 35430548
[23]	Hajipour H, Sambrani R, Ghorbani M, et al. Sildenafil citrate-loaded targeted nanostructured lipid carrier enhances receptivity potential of endometrial cells via LIF and VEGF upregulation[J]. Naunyn Schmiedebergs Arch Pharmacol, 2021, 394(11): 2323-2331.
[24]	Park HR, Choi HJ, Kim BS, et al. Paeoniflorin enhances endometrial receptivity through leukemia inhibitory factor[J]. Biomolecules, 2021, 11(3): 439.
[25]	Bi Y, Huang WY, Yuan LF, et al. HOXA10 improves endometrial receptivity by upregulating E-cadherin[J]. Biol Reprod, 2022, 106(5): 992-999. doi: 10.1093/biolre/ioac007 pmid: 35044439
[26]	Luo HL, Kimura K, Aoki M, et al. Vascular endothelial growth factor(VEGF)promotes the early development of bovine embryo in the presence of cumulus cells[J]. J Vet Med Sci, 2002, 64(11): 967-971.
[27]	Azimi-Nezhad M. Vascular endothelial growth factor from embryonic status to cardiovascular pathology[J]. Rep Biochem Mol Biol, 2014, 2(2): 59-69.
[28]	Wooldridge LK, Ealy AD. Interleukin-6 increases inner cell mass numbers in bovine embryos[J]. BMC Dev Biol, 2019, 19(1): 2.
[29]	Ashary N, Laheri S, Modi D. Homeobox genes in endometrium: from development to decidualization[J]. Int J Dev Biol, 2020, 64(1/2/3): 227-237.
[30]	Gebert LFR, MacRae IJ. Regulation of microRNA function in animals[J]. Nat Rev Mol Cell Biol, 2019, 20(1): 21-37.
[31]	Li Q, Liu WM, Chiu PCN, et al. MiR-let-7a/g enhances uterine receptivity via suppressing Wnt/β-catenin under the modulation of ovarian hormones[J]. Reprod Sci, 2020, 27(5): 1164-1174. doi: 10.1007/s43032-019-00115-3 pmid: 31942710
[32]	Revel A, Achache H, Stevens J, et al. MicroRNAs are associated with human embryo implantation defects[J]. Hum Reprod, 2011, 26(10): 2830-2840.
[33]	Akbar R, Ullah K, Rahman TU, et al. MiR-183-5p regulates uterine receptivity and enhances embryo implantation[J]. J Mol Endocrinol, 2020, 64(1): 43-52. doi: 10.1530/JME-19-0184 pmid: 31786540
[34]	Balaguer N, Moreno I, Herrero M, et al. MicroRNA-30d deficiency during preconception affects endometrial receptivity by decreasing implantation rates and impairing fetal growth[J]. Am J Obstet Gynecol, 2019, 221(1): 46.e1-46.46.e16.
[35]	Kang YJ, Lees M, Matthews LC, et al. MiR-145 suppresses embryo-epithelial juxtacrine communication at implantation by modulating maternal IGF1R[J]. J Cell Sci, 2015, 128(4): 804-814.
[36]	Chen C, Zhao Y, Yu Y, et al. MiR-125b regulates endometrial receptivity by targeting MMP26 in women undergoing IVF-ET with elevated progesterone on HCG priming day[J]. Sci Rep, 2016, 6: 25302. doi: 10.1038/srep25302 pmid: 27143441
[37]	Yu SL, Kang YJ, Jeong DU, et al. The miR-182-5p/NDRG1 axis controls endometrial receptivity through the NF-κB/ZEB1/E-cadherin pathway[J]. Int J Mol Sci, 2022, 23(20): 12303.
[38]	Salilew-Wondim D, Gebremedhn S, Hoelker M, et al. The role of microRNAs in mammalian fertility: from gametogenesis to embryo implantation[J]. Int J Mol Sci, 2020, 21(2): 585.
[39]	Kolanska K, Bendifallah S, Canlorbe G, et al. Role of miRNAs in normal endometrium and in endometrial disorders: comprehensive review[J]. J Clin Med, 2021, 10(16): 3457.