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

Abstract

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

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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References 40

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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

circ-13267 Regulates Egg Duck Granulosa Cells Apoptosis Through Let-7-19/ERBB4 Pathway

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