miR-3604在牛子宫内膜上皮细胞容受性、增殖和凋亡中的作用

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

摘要/Abstract

摘要：

【目的】研究miR-3604在牛子宫内膜上皮细胞（bovines endometrial epithelial cells，bEECs）容受性中的作用。【方法】利用孕酮（progesterone，P4）和干扰素-tau（interferon tau，IFN-τ）联合诱导bEECs构建容受性细胞模型。在此基础上，对容受态bEECs进行牛miR-3604的过表达和干扰后，分别采用RT-qPCR、流式细胞术、EdU和CCK8技术检测了牛miR-3604在bEECs容受性、细胞增殖和凋亡中的作用。【结果】与对照组相比，容受态bEECs的miR-3604过表达组的细胞容受性标志基因HOXA10、IL-6和VEGF的表达量均显著降低（P < 0.05）；细胞凋亡标志基因CASP3和BAX的表达量显著上调（P < 0.05）；容受态bEECs的凋亡率极显著降低（P < 0.01），而细胞增殖率显著升高（P < 0.05）。同时，miR-3604干扰实验的结果与过表达实验的结果相反。【结论】miR-3604在容受性bEECs中的表达量升高，从而可抑制bEECs的容受性和细胞凋亡，并促进bEECs的增殖。

关键词: miR-3604, 子宫内膜容受性, 牛子宫内膜上皮细胞, 细胞增殖, 细胞凋亡

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

姬中祥, 罗仍卓么, 李宇航, 王玉梅, 虎喜敏, 李彦青, 王兴平. miR-3604在牛子宫内膜上皮细胞容受性、增殖和凋亡中的作用[J]. 生物技术通报, 2024, 40(12): 291-298.

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.

图/表 7

表1 RT-qPCR引物信息

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

图1 容受态标志基因及miR-3604在bEECs容受态细胞模型中的表达量 A，B和C：容受性标志基因IL-6、VEGF和LIF的表达量水平；D：miR-3604的表达量水平，*P <0.05，**P <0.01，下同

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

图2 miR-3604在bEECs中的过表达和干扰效率检测 A，B，C和D：miR-3604转染bEECs的荧光观察（100×）；E：mimic转染效率；F：inhibitor转染效率

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

图3 miR-3604对bEECs容受性标志基因表达的影响 A，B和C：miR-3604过表达组VEGF、HOXA10和IL-6的表达量；D，E和F：miR-3604干扰组VEGF、HOXA10和IL-6的表达量

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

图4 miR-3604对容受态bEECs凋亡的影响 A-D： RT-qPCR检测凋亡相关基因CASP3、BAX的表达量；E-J：流式细胞术检测bEECs的凋亡率

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

图5 miR-3604对容受态bEECs增殖的影响 A：增殖细胞荧光观察；B和C：EdU阳性细胞百分比

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

图6 miR-3604对容受态bEECs活力的影响 A：过表达miR-3604对bEECs活力的影响；B：干扰miR-3604对bEECs活力的影响

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

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