马梦楠^,, 王慧明, 王苗苗, 姚望, 张金璧, 潘增祥^,南京农业大学动物科技学院,南京 210095

Identification of circINHBB During Follicular Atresia and Its Effect on Granulosa Cell Apoptosis

MA MengNan^,, WANG HuiMing, WANG MiaoMiao, YAO Wang, ZHANG JinBi, PAN ZengXiang^,College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095

通讯作者: 潘增祥,E-mail： owwa@njau.edu.cn

责任编辑: 林鉴非
收稿日期:2020-06-29接受日期:2021-05-13

基金资助:

国家自然科学基金(31672421) 国家自然科学基金(32002175)

Received:2020-06-29Accepted:2021-05-13
作者简介 About authors
马梦楠,E-mail： 18795975502@163.com。








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.
Keywords：circRNA;circINHBB;follicular atresia;porcine granulosa cell;apoptosis


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本文引用格式
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. 中国农业科学, 2021, 54(18): 3998-4007 doi:10.3864/j.issn.0578-1752.2021.18.017
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. Scientia Acricultura Sinica, 2021, 54(18): 3998-4007 doi:10.3864/j.issn.0578-1752.2021.18.017


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0 引言

【研究意义】卵泡的发育和闭锁关乎哺乳动物的繁殖力,进而影响家畜生产性能。而在哺乳动物中,只有极少数卵泡（少于1%）会被选择并最终排卵为成熟卵泡,而其他卵泡则会发生闭锁^[1]。卵泡闭锁的主要特征是卵母细胞、颗粒细胞和卵泡膜细胞的凋亡,但卵泡闭锁主要诱因是颗粒细胞的凋亡^[2]。在猪卵巢中,细胞凋亡首先发生在壁层颗粒细胞的颗粒层中,而后是卵母细胞和卵丘细胞。在卵泡闭锁过程中,颗粒细胞凋亡增多导致内部容量减少,随着细胞脱落和卵泡腔塌陷,最终使整个卵泡发生退化。近年来,非编码RNA（ncRNA）,尤其是miRNA在卵巢功能中的普遍性和重要功能被逐渐揭开^[3,4,5]。环状RNA(circRNA) 作为一类新发现的内源性ncRNA,可通过几种不同类型的RNA剪接方式以共价键形成环状结构,且不具有5'末端帽子和3'末端,不易被核酸外切酶识别并剪切,因此具备高度的保守性和稳定性。circRNA广泛分布于真核细胞中,参与多种生理过程^[6,7],但其在家畜卵泡中的验证和研究较少。丰富卵泡闭锁过程中circRNA的调控机制的认知,是对家畜繁殖生理的分子调节机理进行补充,会为在实践中提高家畜繁殖力开拓思路和提供有益参考。【前人研究进展】颗粒细胞的凋亡是受到多种细胞因子调控的及其精确和复杂的生理过程,非编码RNA如miRNA已被广泛研究参与颗粒细胞的凋亡^[8,9]。有研究表明circRNA作为调节因子参与了卵巢卵泡的发育过程。例如：在女性卵巢衰老过程中存在差异表达的circRNA,其中circRNA-103827和circRNA-104816参与调控卵巢类固醇的生成,从而影响卵泡的发育^[10]。在成年和幼年的小鼠卵巢中鉴定了许多差异表达的circRNA,其中的circEGFR的表达量在成年小鼠卵巢中显著上调,circEGFR是一种与雌激素信号相关的circRNA,并且circEGFR调控卵泡发育和颗粒细胞的增殖^[11];波尔山羊和麻城黑山羊的卵泡中有37个差异表达的circRNA,并且麻城黑山羊的繁殖率比波尔山羊更高,表明circRNA的差异表达可能对卵巢卵泡的发育产生影响从而导致繁殖率的不同^[12];在牛中,circRNA与BMP15和GDF9相互作用,调节牛卵丘细胞的状态,从而影响卵泡的生长^[13];笔者前期实验通过全基因组circRNA测序证实,猪卵泡中有大量circRNA的表达,且卵泡闭锁过程中的circRNA的表达水平发生了显著变化,表明circRNA在猪卵泡闭锁过程中具有调控潜力。【本研究切入点】抑制素（Inhibin, INH）是一种性腺糖蛋白激素,由两个α亚基或一个α亚基和一个β亚基组成二聚体,主要由雌性动物卵巢卵泡的颗粒细胞产生。INH可以反馈抑制垂体前叶促卵泡激素FSH的释放,调节卵泡的生成,是控制哺乳动物排卵的重要因子^[14]。前期的circRNA测序发现,INHβ亚基的编码基因INHBB可能通过其前体mRNA的反向剪切编码一个circRNA,即circINHBB。但circINHBB的序列结构及其在卵泡闭锁和颗粒细胞中的作用尚待验证和探索。【拟解决的关键问题】本文旨在验证circINHBB的结构和细胞生物学分布,明确其在卵泡闭锁过程中的表达变化,并探索其对颗粒细胞凋亡的调控作用。

1 材料与方法

研究于2018年9月至2019年12月在南京农业大学动物科学类实验教学中心（国家实验教学示范中心）完成。

1.1 样品采集

猪卵巢选自淮安苏食肉品屠宰点180日龄的三元杂交后备母猪,屠宰后10 min内采集表面光滑,卵泡分布均匀,颜色呈淡粉色,无红体与白体的卵巢,置于37℃含双抗（青霉素、链霉素各100 U·mL^-1）的生理盐水中,3 h内带回实验室进行后续试验。

1.2 卵泡的分类

用含有青霉素与链霉素的 37℃ 无菌生理盐水清洗卵巢,在培养皿中用手术刀,眼科镊剥离直径约5mm的单个卵泡。依据外观的形态特征,P4/E2比率和颗粒细胞的密度分为健康卵泡与早期闭锁卵泡,依据参考文献[15]改进的具体标准见表1。

Table 1
表1
表1健康和早期闭锁猪卵泡的判定指标
Table 1Judgment criteria for porcine healthy and early atresia follicles

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

新窗口打开|下载CSV

1.3 颗粒细胞培养

首先用含有青霉素与链霉素的37°C无菌生理盐水和37°C的75%乙醇交替清洗卵巢,然后用10mL注射器抽取直径为3—6mm卵泡的卵泡液及颗粒细胞,1 000×g离心5min去除卵泡液;用含有1%双抗的PBS清洗颗粒细胞两次,1 000×g离心5min去除PBS后,用完全培养基（含体积分数10%胎牛血清和1%双抗的DMEM/F12培养基）重悬细胞,充分吹打至散开并接种于12孔（用于RNA提取和流式细胞实验）或6孔（用于蛋白提取）板中;放置于细胞培养箱内,37℃,5%CO₂培养。36 h后观察颗粒细胞贴壁情况,弃去培养基及未贴壁的颗粒细胞、卵母细胞,用PBS清洗贴壁的颗粒细胞,继续后续试验。

1.4 RNA提取及反转录

猪颗粒细胞转染24h后,用PBS冲洗细胞两次,按照Trizol试剂（Invitrogen公司）说明书提取颗粒细胞RNA（每个孔用量1mL）,用紫外比色法测定总RNA的浓度和纯度,用1.4%甲醛变性琼脂糖凝胶电泳检测总RNA的质量。吸光值为1.8—1.97,且18S、28S条带清晰的RNA样品可用于反转录。取1 μg质量合格的总RNA用PrimeScript RT Master Mix（TaKaRa）试剂盒进行cDNA第一链合成,具体步骤按说明书操作。反转录产物cDNA于-20℃保存备用。

1.5 细胞转染

circINHBB-siRNA, 即si-circINHBB（sens -e：5'-GGCUCAGGCCCCGGCGGAGTT-3'; antisense：5'-CUCCGCCGGGGCCUGAGCCTT-3'）及阴性对照nc-siRNA由吉玛（上海）生物公司合成。 正常培养猪颗粒细胞48h后, 用Lipofectamine 3000 （Invitrogen）和Opti-MEM（Gbico）转染猪颗粒细胞, 继续培养。

1.6 引物设计与qRT-PCR反应

定量引物设计：根据GenBank数据库中收录的INHBB基因mRNA序列和qPCR反应要求,用Primer Premier5.0软件设计反向引物,GAPDH作为内源对照。全部引物由深圳华大生物技术有限公司合成,使用时用ddH₂O稀释至工作浓度。引物序列信息和扩增条件见表2。

Table 2
表2
表2PCR引物及反应条件
Table 2Primer and PCR reaction conditions

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
		R: GCACGGGTCGGTTCAGAAG
GAPDH	AF017079	F: GGACTCATGACCACGGTCCAT	220
		R: TCAGATCCACAACCGACACGT

新窗口打开|下载CSV

PCR的具体操作方法按照2×Vazyme LAmp Master Mix试剂盒（南京诺唯赞）说明进行。PCR产物由琼脂糖凝胶电泳和Sanger测序（上海生工生物工程股份有限公司）进行序列验证。

qRT-PCR的详细步骤参见AceQ qPCR SYBR Green Master Mix试剂盒（南京诺唯赞）说明。GAPDH作为内参。根据LIVAK等^[16]的方法进行数据分析,即处理组的ΔCT=处理组目标基因的CT -处理组内参基因的CT,对照组的ΔCT=对照组目标基因的CT-对照组内参基因的CT;ΔΔCT=处理组的ΔCT -对照组的ΔCT。基因表达的变化倍数表示为2^-ΔΔCT,各基因表达量用均值表示。每个实验组包含6个生物学重复,每个生物学重复进行3次技术重复。

1.7 荧光原位杂交（FISH）

在盖玻片上培养猪颗粒细胞,用4%多聚甲醛（含DEPC）固定20 min,PBS（PH7.3）振荡3次,最后加入蛋白酶K（20 µg·mL^-1）消化5 min。随后用CY3标记的circINHBB特异性探针5'-CGCTCC GCCGG-GGCCTGAGCCCCCG-3'（武汉塞维尔生物公司）标记circINHBB。并用DAPI进行细胞核染色。在Nikon立式荧光显微镜（日本Nikon DS-U3）上采集图像。

1.8 细胞凋亡检测

猪颗粒细胞转染48 h后, 首先用PBS洗涤贴壁的猪颗粒细胞。随后使用Annexin V-FITC/PI staining试剂盒（南京诺唯赞）对颗粒细胞进行染色。最后运用流式细胞仪（FACSCalibur,美BD）检测。使用FlowJo v7.6软件分析数据。每个试验组包含5个技术重复。

1.9 生物信息学软件及在线分析工具

（1）生物信息学软件

Primer premier 5.0 软件：引物设计;

Chromas 2.0软件：对测序结果进行峰图分析;

DNAMAN V5.22 软件：进行核苷酸、氨基酸序列比对分析;

GraphPad Prism 5 软件：绘制图表并进行数据分析。

clipSearch软件：预测circINHBB结合的miRNA。

（2）在线分析工具

GenBank数据（https://www.ncbi.nlm.nih.gov）：获取基因序列;

1.10 统计分析

本实验所有数据分析利用 SPSS 20.0和Prism 5软件进行统计分析。所有实验数据以平均值±标准误（mean±SEM）表示。P<0.05（*）和0.01（**）分别被认为是差异显著和极显著。

2 结果

2.1 健康卵泡与闭锁卵泡的数据

根据课题组前期建立的研究方法,获得直径3—5 mm猪中等卵泡。结合形态学和生物化学标准,即外观形态特征、P4/E2比率和卵泡液颗粒细胞密度指标（表1）区分健康卵泡和早期闭锁卵泡（图1）。依据上述方法选择符合各项判定指标的健康卵泡6个、早期闭锁卵泡6个。样本信息及具体分组见表3。

图1

新窗口打开|下载原图ZIP|生成PPT
图1健康卵泡（H）与早期闭锁猪卵泡（EA）的外观形态

Fig. 1Appearance of porcine healthy (H) and early atresia (EA) follicles



Table 3
表3
表3卵泡样本信息
Table 3Sample information of ovary

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

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2.2 circRNA的鉴定

由于circRNA由mRNA前体反向剪切形成,设计引物时以预测的线性RNA剪切位点方向为引物3′端,向线性RNA两侧方向设计引物,从而扩增包含剪切位点的circRNA序列。circINHBB的正、反向引物设计原理如图2-A所示。circINHBB的PCR产物电泳结果显示出单一条带,表明了circINHBB在卵泡中有表达,且具有特异性（图2-B）。

图2

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图2circINHBB的鉴定

（A）circRNA引物设计原理图;（B）circINHBB的PCR产物电泳图;（C）circINHBB的Sanger序列测序峰图及circINHBB剪接点
Fig. 2Identification of circINHBB

(A) Schematic diagram of circRNA primer design; (B) Electrophoresis analysis of circINHBB PCR product; (C) Electropherogram of circINHBB Sanger sequencing and splicing site of circINHBB


对PCR产物进行Sanger测序的结果进一步确认了circINHBB的碱基序列,并明确了INHBB 线性mRNA前体（5′UTR区域）成环反应的剪切位点（图2-C）。

2.3 circINHBB在猪颗粒细胞中的分布

为了验证circINHBB在猪卵泡颗粒细胞中的分布情况,我们通过FISH方法,用circINHBB探针检测了其在健康颗粒细胞中的表达情况。结果可见circINHBB（红色荧光）主要存在于颗粒细胞质中,且表达量较高,在细胞核中表达量较低（图3）。由此可见circINHBB可能主要通过转录后机制参与对颗粒细胞的调控。

图3

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图3circINHBB的细胞定位

红色荧光探针标记 circINHBB,蓝色DAPI标记细胞核;标尺长度为50μm
Fig. 3The localization of circINHA detected by FISH

CircINHA was labelled by red fluorescence, Nuclei were stained by DAPI. Scale bar, 50 μm

2.4 circINHBB在猪健康、早期闭锁卵泡的差异表达

对健康和早期闭锁卵泡的circINHBB进行qRT- PCR检测可见circINHBB在健康卵泡中表达量较高,而在早期闭锁卵泡中表达量显著下降（图4）,可见circINHBB参与了卵泡闭锁过程。

图4

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图4circINHBB在健康和早期闭锁卵泡中的表达

“*”表示 P<0.05
Fig. 4circINHBB expression in healthy and early atrestic follicles

”*” means P<0.05

2.5 circINHBB抑制猪颗粒细胞的凋亡

为了探索circINHBB对猪颗粒细胞凋亡的影响,我们首先合成了circINHBB的特异性siRNA,然后将其转染到猪颗粒细胞中。qRT-PCR结果证明si-circINHBB能显著抑制circINHBB的表达水平,敲减效率约为50%（图5-A）。同时INHBB的线性mRNA表达水平不受si-circINHBB的影响（图5-B）,可见该siRNA的敲减效果具有特异性。随后,用流式细胞术（FACS）检测si-circINHBB转染前后的颗粒细胞凋亡水平。结果显示,敲减circINHBB后,颗粒细胞凋亡率显著上升,可见circINHBB对颗粒细胞凋亡有显著的抑制作用（图5-C）。

图5

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图5敲减circINHBB 促进猪颗粒细胞凋亡

A：si-circINHBB有效敲减circINHBB;B：si-circINHBB对INHBB mRNA表达量没有影响;C：si-circINHBB促进猪颗粒细胞凋亡
Fig. 5Knockdown of circINHBB promotes apoptosis of porcine granulosa cells

A: circINHBB was knockdown by si-circINHBB effectively; b: si-circINHBB had no effect on INHBB mRNA expression; c: si-circINHBB promoted the apoptosis of porcine granulosa cells

2.6 circINHBB互作的miRNA

circINHBB存在于猪卵泡颗粒细胞的细胞质中,提示其可能作为miRNA的分子海绵调控猪卵泡的发育。为进一步探索circINHB可能参与调节的miRNA,我们利用生物学信息工具预测circINHBB互作的miRNA,结果表明,circINHBB可能与10个miRNA相互作用（表4）。

Table 4
表4
表4circINHBB互作的miRNA
Table 4circINHBB interacting miRNA

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

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3 讨论

近年来,circRNA参与的分子调控成为了ncRNA调控研究的新热点。circRNA对核酸外切酶具有抗性,因此在哺乳动物细胞中半衰期较长。circRNAs的普遍性、保守性、组织/细胞特异性和稳定性使其成为了比miRNA更理想的生物标志物候选者^[17]。研究表明在人类血液、唾液和胃液中也检测到了circRNAs,这进一步提高了circRNAs作为有效诊断和预后生物标志物的潜力^[18]。目前,在人卵巢颗粒细胞^[19]、胎盘^[20]和睾丸^[21]中的circRNA表达谱已有研究,并得出了circRNA与这些生殖相关组织的病理特征密切相关的结论。笔者前期在猪中等有腔卵泡中的circRNA表达谱研究^[22]是家畜繁殖学领域中对circRNA的首次探索。本研究验证了circINHBB在猪卵泡颗粒细胞中的表达,并发现其与卵泡闭锁过程息息相关。卵泡中的circINHBB的鉴定可能为畜牧业分子育种和生殖医学提供新的circRNA生物标记物。最近,越来越多的证据表明,circRNA可能通过多作为竞争性内源性RNA（ceRNA）,通过高效结合某个miRNA^[23]或作为miRNAs海绵^[24]特异性吸附多个miRNAs,从转录后水平调节基因的表达。笔者研究发现circINHBB主要在颗粒细胞质中表达。表明circINHBB可能是通过作为miRNAs分子海绵发挥生物学功能。

在生殖领域,miRNA在颗粒细胞凋亡、卵泡闭锁过程中的参与已经在人、小鼠、猪和牛身上得到了很好的研究和广泛证实^[18]。笔者通过生物信息工具的预测分析发现,circINHBB可能与10个miRNA相互作用。研究表明,miR-342是Notch信号通路的下游分子,miR-342可以调控TGF-β信号通路^[25]并且miR- 342抑制PIK3R1的表达从而激活Akt信号通路^[26];miR-432通过抑制IGF的表达抑制Akt信号通路从而抑制细胞的增殖^[27];miR-122可以通过直接靶向TGFBR2抑制TGF-β/ Smad信号通路^[28]。大量的研究表明TGF-β信号通路调控猪卵泡的发育和颗粒细胞的凋亡^[29,30,31];Notch信号通路调控卵泡的生长,卵母细胞的减数分裂,卵巢的血管生成和类固醇激素的生成^[32];在猪卵泡颗粒细胞中,抑制Notch信号通路时,将促进NPC1和StAR的表达从而刺激孕激素分泌^[33];此外,研究证实IGF通过PI3K/ Akt信号通路促进猪颗粒细胞的增殖^[34]。综上,circINHBB可能通过竞争结合miR-342、miR-432和miR-122从而调控卵泡的闭锁（图6）。

图6

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图6circINHBB可能参与的调控机制

circINHBB可能通过①与线性mRNA竞争性剪接;或②特异性结合miRNA两种方式参与基因表达调控
Fig. 6The regulation mechanism that circINHBB may be involved in

circINHBB may participate in gene expression regulation by ① competitive splicing with linear mRNA or ② specific binding to miRNAs


值得注意的是,INHBB作为circINHBB的编码基因,同时也编码INHB的β亚基,而INH是重要的FSH的负调节因子。据报道,抑制素活性的降低可能导致FSH水平升高,随后每个周期招募的卵泡数量过多,最终导致卵泡池提前耗尽,造成卵巢早衰（POF）^[14]。circINHBB的剪接可能通过与线性INHBB mRNA的竞争,抑制INHBB的剪接和翻译,从而促进FSH的调节作用,促进颗粒细胞增殖以及卵泡的生长和发育（图6）。

4 结论

笔者在猪卵泡中鉴定了一个新的,由INHBB基因编码的circRNA,即circINHBB。它在卵泡颗粒细胞质中表达,在猪卵泡闭锁过程中显著下调,并具有抑制颗粒细胞凋亡的功能。circINHBB的作用机制可能通过作为miRNA的分子海绵,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。本研究为进一步完善circRNA对卵泡闭锁、颗粒细胞凋亡的影响及调控机理提供了参考。

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