circ-13267通过let-7-19/ERBB4通路调控蛋鸭卵泡颗粒细胞凋亡

doi:10.3864/j.issn.0578-1752.2022.08.015

摘要/Abstract

摘要：

【背景】蛋鸭卵泡发育是决定其产蛋性能的关键因素。已有研究表明,禽类卵泡发育是极为复杂的生物学过程,目前人们已对家禽的卵泡发育模式有了一定的了解,但作为决定产蛋量的重要因素,卵泡发育的具体调控机理仍需进一步深入研究。颗粒细胞是卵泡中主要的功能细胞,可调控卵泡膜细胞和卵母细胞的生长、分化和成熟,同时也精确的调控卵泡的生长发育,维持卵巢的正常功能（诱导排卵、维持成熟分裂的阻断、为卵母细胞提供底物等）。circRNA是一类新型的内源性非编码RNA,在卵泡发育中发挥重要的调控作用。【目的】通过构建circRNA过表达载体上调circ-13267的表达,研究circ-13267在蛋鸭卵泡颗粒细胞中的作用及其调控机制,为蛋鸭卵泡发育的调控机制解析提供依据。【方法】首先,利用Q-PCR检测蛋鸭卵泡颗粒细胞的细胞质与细胞核中circ-13267的表达水平;构建circ-13267的过表达载体circ-13267-pLCDH,在蛋鸭颗粒细胞中过表达circ-13267后,利用Q-PCR法检测circ-13267、let-7-19、ERBB4、FAS和BCL2的表达水平;分别转染circ-13267-pLCDH和pLCDH-ciR于蛋鸭卵泡颗粒细胞,24 h后利用EdU法检测蛋鸭卵泡颗粒细胞的增殖能力;将circ-13267的线性序列或ERBB4的3′ UTR克隆到pmirGLo载体中,同时对上述野生型序列中的let-7-19结合位点进行突变,得到表达突变型序列的载体,利用双荧光素酶报告基因验证circ-13267与let-7-19及let-7-19与靶基因ERBB4的结合关系;在蛋鸭卵泡颗粒细胞中转染circ-13267-pLCDH和pLCDH-ciR,利用流式细胞术和Annexin V-FITC检测蛋鸭卵泡颗粒细胞凋亡情况。【结果】环状RNA circ-13267在细胞质和细胞核中均有表达;双荧光素酶报告基因试验结果显示,let-7-19能与ERBB4结合,进而下调荧光素酶的活性,当ERBB4序列中let-7-19的结合位点突变后,let-7-19则无法抑制荧光素酶的表达,说明ERBB4是let-7-19的一个靶基因;荧光定量检测结果显示,过表达环状RNAcirc-13267后BCL2基因的表达显著降低（P<0.05）,而FAS和ERBB4基因的表达量显著升高（P<0.05）,当过表达let-7-19后,ERBB4基因的表达量显著升高（P<0.05）,而抑制let-7-19后ERBB4基因的表达量显著降低（P<0.05）;EdU试验结果显示,过表达circ-13267后蛋鸭卵泡颗粒细胞数量显著减少,说明其促进了蛋鸭卵泡颗粒细胞的凋亡;然而,在蛋鸭卵泡颗粒细胞中共转染circ-13267和let-7-19后,与对照组相比共转染组中BCL2和FAS的表达量均无显著变化（P>0.05）,而与过表达circ-13267组相比,共转组中BCL2基因的表达量极显著降低（P<0.01）、FAS的表达量极显著升高（P<0.01）,说明circ-13267促进蛋鸭卵泡颗粒细胞凋亡的作用被抑制;利用流式细胞仪检测转染后的蛋鸭卵泡颗粒细胞发现,与共转染circ-13267和let-7-19组相比,仅过表达circ-13267后,晚期凋亡细胞数和总凋亡细胞数均显著增加（P<0.05）,而活细胞数显著降低（P<0.05）。【结论】蛋鸭circ-13267在蛋鸭卵泡颗粒细胞的细胞质和细胞核中均有表达,circ-13267可以吸附let-7-19并靶向ERBB4基因,从而促进了蛋鸭卵泡颗粒细胞的凋亡,为解析蛋鸭卵泡发育的调控机制提供理论依据。

关键词: circRNA, miRNA, 蛋鸭, 卵泡发育, 颗粒细胞

Abstract:

【Background】 Follicle development is a key factor for laying performance of egg ducks. Previous studies have shown that follicular development is a very complex biological process in poultry. At present, the pattern of follicular development in poultry has been understood. However, as an important factor determining egg production, the specific regulation mechanism of follicular development still needs further study. Granulosa cells are the main functional cells in follicles. They can regulate the growth, differentiation and maturation of theca cells and oocytes. They also regulate the growth and development of follicles, maintain normal ovarian function, such as induce ovulation, maintain maturation division, and provide substrates for oocytes. Circular RNAs (circRNAs) are a new type of endogenous specific non-coding RNA, which plays an important role in follicular development. 【Objective】The objective of this study was to explore the effects and regulatory mechanism of circ-13267 on apoptosis in egg duck granulosa cells, through regulating the expression of circ-13267 by constructing the overexpression vector, so as to provide the evidence for analysis the regulatory mechanism of egg duck follicular development. 【Method】Firstly, the expression levels of circ-13267 in cytoplasm and nucleus of granulosa cells was detected by Q-PCR. The overexpression vector circ-13267-pLCDH was constructed. After transfection of circ-13267 in egg duck granulosa cells, the expression levels of circ-13267, let-7-19, ERBB4, FAS and BCL2 were detected by Q-PCR. The proliferation of egg duck granulosa cells was detected by EdU method after transfection circ-13267-pLCDH and pLCDH-ciR. The linear sequence of circ-13267 or the 3'UTR of ErbB4 was cloned into pmirGLo vector. At the same time, let-7-19 binding site in the wild-type sequence was mutated to obtain the vector expressing the mutant sequence. The targeting relationships between circ-13267 and let-7-19, let-7-19 and ERBB4 were verified by dual luciferase reporter assay, respectively. Then, after transfection of circ-13267-pLCDH and pLCDH-ciR into egg duck follicular granulosa cells, the flow cytometry and Annexin V-FITC were utilized to explore the effects of circ-13267 on duck granulosa cells. 【Result】 In duck granulosa cells, circ-13267 was expressed in both cytoplasm and nucleus. The dual luciferase reporter gene assay confirmed that let-7-19 could bind to ERBB4 and down regulate the activity of luciferase; when the binding site of let-7-19 in ErbB4 sequence was mutated, let-7-19 could not inhibit the expression of luciferase, indicating that ERBB4 was a target gene of let-7-19. The results of Q-PCR showed that, after overexpression of circ-13267, the expression of BCL2 gene decreased significantly (P<0.05), while the expression of FAS and ERBB4 gene increased significantly (P<0.05); after overexpression of let-7-19, the expression of ERBB4 gene increased significantly (P<0.05), while after inhibition of let-7-19, the expression of ERBB4 gene decreased significantly (P<0.05). EdU test results showed that the number of follicular granulosa cells in egg ducks decreased significantly after overexpression of circ-13267, it was shown that circ-13267 promoted the apoptosis of follicular granulosa cells in egg ducks. However, after co-transfection of circ-13267 and let-7-19 into egg duck follicular granulosa cells, compared with the control group, there was no significant change in the expression of BCL2 and FAS (P>0.05); however, compared with overexpression of circ-13267, the expression of BCL2 gene decreased significantly (P<0.01) and FAS increased significantly (P<0.01). It was shown that circ-13267 could inhibit the apoptosis of egg duck follicular granulosa cells. In addition, flow cytometry was used to detect the transfected egg duck follicular granulosa cells. Compared with the co-transfection groups of circ-13267 and let-7-19, the number of late apoptotic cells and total apoptotic cells increased significantly (P<0.05), while the number of living cells decreased significantly (P<0.05). 【Conclusion】 circ-13267 was expressed in the cytoplasm and nucleus of egg duck follicular granulosa cells. circ-13267 could sponge let-7-19 and target ERBB4 gene, which promoted the apoptosis of egg duck follicular granulosa cells. This results provided a theoretical basis for analysis of the regulatory mechanism of egg duck follicular development.

Key words: circRNA, miRNA, egg duck, follicular development, granulosa cells

吴艳,张昊,梁振华,潘爱銮,申杰,蒲跃进,黄涛,皮劲松,杜金平. circ-13267通过let-7-19/ERBB4通路调控蛋鸭卵泡颗粒细胞凋亡[J]. 中国农业科学, 2022, 55(8): 1657-1666.

WU Yan,ZHANG Hao,LIANG ZhenHua,PAN AiLuan,SHEN Jie,PU YueJin,HUANG Tao,PI JinSong,DU JinPing. circ-13267 Regulates Egg Duck Granulosa Cells Apoptosis Through Let-7-19/ERBB4 Pathway[J]. Scientia Agricultura Sinica, 2022, 55(8): 1657-1666.

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引物名称 Primer name	引物序列 Primer sequence	产物长度 Product length (bp)
circ_13267	F:CACTAAAATAAAATCTGTTCAATTAACGAATTCGAGGAAACCCCTATGGTAACATC R:GGCGTTATCATCCCAAATTAGTGGATCCTGGAAGGGCTCATGAAATCTTAC	1488
circ_13267-q	F: CGGAATTTCCTCCAACTGA R: CTGCTGCCTGGTCTGTTAC	104
β-actin	F: CTCGCTTCGGCAGCACA R: AACGCTTCACGAATTTGCGT	201
let-7-19	F: ACACTCCAGCTGGGTGAGGTAGTAGTTTGTG LOOP: CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGACAGCAC URP: TGGTGTCGTGGAGTCG	-
U6	F: CTCGCTTCGGCAGCACA R: AACGCTTCACGAATTTGCGT	-
FAS	F: GCCCCAGTTGAAGAAAAA R: TCCAGGCAAGTAAGACCC	171
BCL2	F: ACGGCTCTCG CTCCTGCT R: CGGTTGACGC TCTCCACG	260
ERBB4	F: ATTTCAGGGTGTTTTCTA R: TCTGCTTTTTTATTTCGG	244