猪卵泡闭锁过程中circINHBB的鉴定及其对颗粒细胞凋亡的影响

doi:10.3864/j.issn.0578-1752.2021.18.017

摘要/Abstract

摘要：

【背景】卵泡的发育状况和成熟排卵数量是哺乳动物的繁殖力和生产性能的重要决定因素。卵泡闭锁是卵泡停止发育并发生退化的过程,可能发生在卵泡发育的各个阶段,而闭锁的发生与卵泡颗粒细胞的增殖和凋亡情况密切相关,而颗粒细胞的凋亡过程十分复杂并且受到各种细胞因子的调控作用。环状RNA（circRNA）是近年来在生物体内发现的一种环状结构非编码RNAs（ncRNAs）。研究证明circRNA普遍存在于各个组织且参与到各种生理过程的调节中,但其在家畜繁殖学领域,尤其是猪卵巢和卵泡中的表达变化、位置分布和生物学功能研究较少。抑制素（INH）是一种性腺糖蛋白激素,主要由雌性动物卵巢卵泡的颗粒细胞产生,是控制哺乳动物排卵的重要因子。前期实验发现,猪卵泡中INHβ亚基INHBB编码基因的mRNA的前体可能形成一个circRNA,即circINHBB。【目的】在猪中等有腔卵泡组织中验证circINHBB的序列结构及其在颗粒细胞中的分布情况;分析其在健康、闭锁卵泡中的表达差异;在体外培养的猪卵泡颗粒细胞中探索了circINHBB对细胞凋亡的调控作用,并对circINHBB可能介导的功能性miRNA做出预测,为家畜繁殖领域的circRNAs研究扩宽思路,为提高家畜繁殖力研究提供参考。【方法】采集中等大小猪卵泡,进行RNA提取及反转录获得猪卵泡的cDNA,利用反向引物进行PCR扩增,PCR扩增产物经Sanger测序证明circINHBB的序列和环状结构;设计circINHBB的特异性荧光探针,运用FISH实验验证circINHBB在猪卵泡颗粒细胞核、质中的分布情况;然后,通过外观、激素和颗粒细胞密度指标选取健康和闭锁两组,用qRT-PCR检测circINHBB在猪健康和闭锁卵泡中的表达差异;最后,设计circINHBB的特异性siRNA（si-circINHBB）,在体外培养的猪卵泡颗粒细胞中,转染si-circINHBB及其对照,利用流式细胞术检测circINHBB对猪卵泡颗粒细胞凋亡的调控作用,预测circRNA可能参与的miRNA调节。【结果】PCR及Sanger测序验证了circINHBB在猪卵泡中的特异性存在,且证实了circINHBB是由INHBB编码基因的mRNA的前体经反向可变剪切形成的环状结构RNA;FISH实验进一步验证了circINHBB在猪卵泡颗粒细胞的细胞质中分布;qRT-PCR结果证实,与猪健康卵泡相比,circINHBB的表达量在猪闭锁卵泡中显著降低;流式细胞术检测结果表明当敲减circINHBB后,猪卵泡颗粒细胞的凋亡水平显著上升,说明circINHBB对猪卵泡颗粒细胞的凋亡有显著的抑制作用;生物信息学分析表明,circINHBB可能与10个已知miRNA相互作用,通过TGF-β、Notch等信号通路参与调控颗粒细胞凋亡及卵泡闭锁过程。【结论】在猪卵泡中验证了circINHBB的环状结构及胞质中的特异性表达分布,证实了其在闭锁卵泡中表达量低于健康卵泡,通过体外实验证实了circINHBB是细胞凋亡的抑制因子,可能通过吸附相关miRNA参与调节卵泡的发育和闭锁过程。

关键词: circRNA, circINHBB, 卵泡闭锁, 猪颗粒细胞, 凋亡

Abstract:

【Background】The follicular development is related to the reproductive capacity and production performance of mammals. Follicular development includes the growth and development of oocytes, the initiation and growth of the primordial follicles, and the development of primary follicles. In addition, these biological processes are closely related to the proliferation and apoptosis processes of follicle granulosa cells. The main inducement of follicular atresia is the apoptosis of granulosa cells, which is extremely complex and regulated by various cytokines. Previous studies have shown that non-coding RNAs (ncRNAs) are involved in various biological processes as regulators, including cell proliferation and apoptosis. Circular RNA (circRNA) is a new type of ncRNAs, widely existing in organisms. The circRNA is involved in the regulation of various physiological processes, but there are few studies in the field of livestock reproduction, especially, its expression, distribution and biological function in the pig ovary and follicle are rarely studied. Inhibin (INH) is a gonadal glycoprotein hormone, which is mainly produced by granulosa cells of ovarian follicles in female animals. It is an important factor to control ovulation in mammals. Previous studies have shown that the coding gene of the precursor of INH βsubunit in porcine follicles may form a circular RNA, i.e. circINHBB. 【Objective】In this study, the sequence structure and cell distribution of circINHBB in porcine follicular tissue were verified, and its expression difference in healthy and atresia follicles analyzed to explore the effect of circINHBB on cell apoptosis in porcine granulosa cells cultured in vitro, so as to broaden the research ideas of circRNAs in the field of livestock breeding, and provide reference for improving the reproductive capacity of livestock. 【Method】 Firstly, the porcine follicles were collected, amplified by PCR and sequenced by Sanger sequencing to verify the sequence structure of circINHBB. Secondly, the follicles were divided into two groups, including healthy and atretic, and the expression difference of circINHBB was detected by qRT PCR in each group. Then, the FISH experiment was used to verify the distribution of circINHBB in the granulosa cells. Finally, the porcine ovarian granulosa cells were cultured in vitro, circINHBB was knocked down by siRNA, and the effect of circINHBB on granulosa cell apoptosis was detected by flow cytometry. 【Result】Sanger sequencing confirmed the existence of circINHBB in porcine follicles, and it was a circular RNA formed by reverse splicing of the INHBB mRNA 5’UTR. qRT PCR results confirmed that the expression level of circINHBB was higher in healthy follicles, but significantly decreased in atresia follicles. FISH further verified the distribution of circINHBB in the cytoplasm of porcine granulosa cells. After siRNA knockdown, the expression of circINHBB was significantly decreased, and the apoptosis rate of granulosa cells increased significantly. 【Conclusion】The circular structure of circINHBB was verified in the porcine ovary, and its distribution in cytoplasm suggested that circINHBB might be involved in post-transcriptional regulation. The fact that the expression of circINHBB was lower in atresia follicles, and knockdown of circINHBB could significantly increase the level of apoptosis, indicating that circINHBB had a significant inhibitory effect on the apoptosis of porcine granulosa cells. Thus, it might be an active regulatory factor in the growth and development of follicles. This study explored the role of circRNA in follicular atresia and granulosa cell apoptosis, which was an important supplement to the regulatory mechanism of follicular atresia.

Key words: circRNA, circINHBB, follicular atresia, porcine granulosa cell, apoptosis

马梦楠,王慧明,王苗苗,姚望,张金璧,潘增祥. 猪卵泡闭锁过程中circINHBB的鉴定及其对颗粒细胞凋亡的影响[J]. 中国农业科学, 2021, 54(18): 3998-4007.

MA MengNan,WANG HuiMing,WANG MiaoMiao,YAO Wang,ZHANG JinBi,PAN ZengXiang. Identification of circINHBB During Follicular Atresia and Its Effect on Granulosa Cell Apoptosis[J]. Scientia Agricultura Sinica, 2021, 54(18): 3998-4007.

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卵泡类型 Follicle type	孕酮/雌二醇 P4/E2	颗粒细胞密度 GC density（cells/µL）	大小 Size（mm）	颜色 Colour	通透度 Clarity	血管数量 Number of blood vessels
健康卵泡 HF	≤1.3	≤25000	4-5	红 Red	透亮Transparent	多 Many
早期闭锁卵泡 EA	≥1.8	≥50000	4-5	黄/白Yellow/White	浑浊 Turbid	少 Less

基因 Gene	登录号 Acc.No.	引物(5'- 3') Primer (5'- 3')	产物长度 Product length（bp）
circINHBB	AY116585.1	F: AGGGCGAGACGCTCAGGT	223
circINHBB	AY116585.1	R: GCACGGGTCGGTTCAGAAG	223
GAPDH	AF017079	F: GGACTCATGACCACGGTCCAT	220
GAPDH	AF017079	R: TCAGATCCACAACCGACACGT	220

样品编号 Sample No.	孕酮/雌二醇 P4/E2	颗粒细胞密度 GC density (cells/μL)	颜色 Colour	通透度 Clarity	血管数量 Number of blood vessels
H1	0.30	22000	红 Red	透亮 Transparent	多 Many
H2	0.66	20250	红 Red	透亮 Transparent	多 Many
H3	0.95	19500	红 Red	透亮 Transparent	多 Many
H4	1.09	19750	红 Red	透亮 Transparent	多 Many
H5	1.22	14000	红 Red	透亮 Transparent	多 Many
H6	1.29	16500	红 Red	透亮 Transparent	多 Many
EA1	1.96	50000	黄 Yellow	浑浊 Turbid	少 Less
EA2	2.13	50000	白 White	浑浊 Turbid	少 Less
EA3	3.72	62500	黄 Yellow	浑浊 Turbid	少 Less
EA4	5.39	62500	黄 Yellow	浑浊 Turbid	少 Less
EA5	8.66	100000	白 White	浑浊 Turbid	少 Less
EA6	16.20	75000	白 White	浑浊 Turbid	少 Less

结合的miRNA Bound miRNA	结合类型 Combination type	自由能 Free Energy	分数 Fraction
ssc-miR-10391	7mer-m8	-20.4	36
ssc-miR-342	7mer-m8	-16.9	32
ssc-miR-432-5p	7mer-m8	-18.8	38
ssc-miR-744	7mer-m8	-28.0	39
ssc-miR-9810-3p	7mer-m8	-12.4	24
ssc-miR-9824-5p	7mer-m8	-20.8	33
ssc-miR-122-5p	7mer-A1	-14.8	33
ssc-miR-1249	7mer-A1	-16.7	26
ssc-miR-181d-3p	7mer-A1	-13.7	18
ssc-miR-2411	7mer-A1	-12.9	33