干扰KISS1基因对猪卵巢颗粒细胞功能的影响

doi:10.3864/j.issn.0578-1752.2020.23.018

中国农业科学 ›› 2020, Vol. 53 ›› Issue (23): 4940-4949.doi: 10.3864/j.issn.0578-1752.2020.23.018

• 畜牧·兽医·资源昆虫 • 上一篇下一篇

干扰KISS1基因对猪卵巢颗粒细胞功能的影响

陆思羽¹(),何颖婷¹,周小枫¹,辛晓萍¹,张爱玲²,袁晓龙¹,张哲¹,李加琪¹()

¹广东省农业动物基因组学与分子育种重点实验室/国家生猪种业工程技术研究中心/华南农业大学动物科学学院,广州510642
²广东高校应用生态工程技术开发中心/广东第二师范学院生物与食品工程学院,广州510310

收稿日期:2019-11-28 接受日期:2020-10-29 出版日期:2020-12-01 发布日期:2020-12-09
通讯作者: 李加琪
作者简介:陆思羽,E-mail： siyu_lu19@163.com。
基金资助:
国家生猪现代农业产业技术体系(CARS-35);国家自然科学基金青年项目(31902131);广东省自然科学基金面上项目(2019A1515010676);广东省扬帆计划(2014YT02H042);广东省普通高校青年创新人才项目(2018KQNCX019);广州市科技计划项目(201707010001)

Effect of KISS1 Interference on the Function of Porcine Granulosa Cells in Porcine Ovary

LU SiYu¹(),HE YingTing¹,ZHOU XiaoFeng¹,XIN XiaoPing¹,ZHANG AiLing²,YUAN XiaoLong¹,ZHANG Zhe¹,LI JiaQi¹()

¹Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding/National Engineering Research Center for Breeding Swine Industry/College of Animal Science, South China Agricultural University, Guangzhou 510642
²Guangdong Development Center of Applied Ecology and Ecological Engineering in Universities/Biology and Food Engineering Institute,Guangdong University of Education, Guangzhou 510310

Received:2019-11-28 Accepted:2020-10-29 Online:2020-12-01 Published:2020-12-09
Contact: JiaQi LI

摘要/Abstract

摘要：

【背景】卵泡是卵巢的基本结构和功能单位,其主要功能是排卵和分泌激素。颗粒细胞能促进卵泡发育,其过度凋亡能抑制卵泡发育,诱导卵泡闭锁,进而降低雌性动物发情频率,影响雌性动物繁殖力。现已有研究发现,KISS1在卵巢组织中发挥着重要作用。【目的】研究通过干扰KISS1,以阐释KISS1对猪卵巢颗粒细胞凋亡、周期及分泌雌激素能力的影响,为完善KISS1在猪颗粒细胞中的分子调控机制提供一定的依据。【方法】设计KISS1的干扰片段KISS1-siRNA,转染体外培养的母猪卵巢颗粒细胞,通过实时定量PCR（quantitative real time PCR, qRT-PCR）检测干扰KISS1对母猪卵巢颗粒细胞中磷脂酰肌醇3-激酶（phosphoinositide 3-kinase, PI3K）信号通路部分基因转录水平的影响;采用流式检测法、Annexin V- FITC及ELISA技术,分别探究干扰KISS1对颗粒细胞周期、凋亡及雌二醇（estradiol, E₂）分泌量的影响,最后使用qRT-PCR技术检测KISS1对雌激素受体及雌激素信号通路关键基因转录水平的影响。【结果】在猪颗粒细胞内,干扰KISS1后,PI3K通路激活相关基因PIK3CG、PI3CI、PDK1及AKT1转录水平下降,其中关键基因AKT1的转录水平显著降低（P<0.05）,PI3K通路激活抑制相关基因FOXO3、TSC2及BAD转录水平也有所降低;干扰KISS1后,颗粒细胞周期在细胞分裂间期（G0/ G1）阻断,细胞的凋亡率显著上升,细胞中E₂的浓度显著降低（P<0.01）,雌激素受体ESR1和ESR2及雌激素通路的基因Star、CYP17、3B-HSD、17B-HSD和CYP19A转录水平也相应显著下降（P<0.05）。【结论】KISS1能够参与猪颗粒细胞PI3K和雌激素通路,干扰KISS1能够使卵巢颗粒细胞阻滞在细胞分裂间期,促进颗粒细胞凋亡,降低颗粒细胞分泌雌激素的能力,表明KISS1对于卵巢颗粒细胞的分裂与生长、雌激素分泌具有重要作用。

关键词: 母猪, 颗粒细胞, KISS1, PI3K, E₂

Abstract:

【Background】 As the basic structural and functional unit of ovary, the main functions of follicles are ovulation and hormone secretion. Granulosa cells proliferation can promote the development of follicles, while the excessive apoptosis of granulosa cells will inhibit the development of follicles, induce follicular atresia, reduce the estrus frequency and affect the reproductive capacity of female animals. Recent studies have found that the KISS1 plays an important role in ovary. 【Objective】 This study was aimed to explore the effects of KISS1 on apoptosis, cell cycle and estrogen secretion of porcine granulosa cells, through interfering the expression of KISS1, so as to provide some useful information in molecular regulation mechanism of KISS1 on porcine granulosa cells. 【Method】 To investigate the effect of knockdown KISS1 on the transcription of key genes on phosphatidylinositol 3-kinase (PI3K) signaling pathway in porcine granulosa cells, KISS1-siRNA were designed and transfected to porcine granulosa cells in vitro, and then quantitative real time PCR (qRT PCR) was applied to detect the transcription of KISS1. Then, flow cytometry, Annexin V-FITC, and ELISA were utilized to explore the effects of KISS1-siRNA on granulosa cell cycle, apoptosis, and secretion of E₂, respectively. Finally, qRT-PCR was applied to explore the effects of KISS1-siRNA on estrogen receptor and estrogen signaling pathway. 【Result】 In porcine granulosa cells, interfering KISS1 gene could significantly decrease the transcription of AKT1(P<0.05), while PIK3CG, PI3CI and PDK1 decreased with no significant difference (P>0.05). The mRNA levels of the genes, involving in activation of PI3K signaling pathway like FOXO3, TSC2, and BAD, were also decreased with no significant difference (P>0.05). Transfecting KISS1-siRNA to porcine granulosa cells could significantly promote cell apoptosis (P<0.01), arrest cell cycle at G0/G1 phase (P<0.01), and decrease the E₂ secretion (P<0.01). After knockdown KISS1, the transcription of estrogen receptor ESR1 and ESR2, as well as Star, CYP17, 3B-HSD, 17B-HSD and CYP19A of estrogen pathway were also decreased significantly (P<0.05). 【Conclusion】 This study confirmed that KISS1 gene could participate in PI3K and estrogen signaling pathways. Interfering KISS1 could promote porcine granulosa cells apoptosis, arrest cell cycle at G0/G1 phase (P<0.01), and decrease the E₂ secretion. It suggested that KISS1 played an important role in the division, growth and estrogen secretion of porcine granulosa cells.

Key words: sows, granulosa cells, KISS1, PI3K, E₂

陆思羽,何颖婷,周小枫,辛晓萍,张爱玲,袁晓龙,张哲,李加琪. 干扰KISS1基因对猪卵巢颗粒细胞功能的影响[J]. 中国农业科学, 2020, 53(23): 4940-4949.

LU SiYu,HE YingTing,ZHOU XiaoFeng,XIN XiaoPing,ZHANG AiLing,YUAN XiaoLong,ZHANG Zhe,LI JiaQi. Effect of KISS1 Interference on the Function of Porcine Granulosa Cells in Porcine Ovary[J]. Scientia Agricultura Sinica, 2020, 53(23): 4940-4949.

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参考文献 41

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引物名称 Primer name	Gene ID	引物序列 Primer sequence	产物大小 Size (bp)
qRT-KISS1	100145896	F: AACCAGCATCTTCTCACCAGG	192
qRT-KISS1	100145896	R: CTTTCTCTCCGCACAACGC	192
qRT-PIK3CG	396979	F: AACGGGCTTTGAGATAGTGAA	184
qRT-PIK3CG	396979	R: AAGTTGCTTGGTTGGTGGATA	184
qRT-PIK3C1	5290	F: CAAGTGAGAATGGTCCGAATG	152
qRT-PIK3C1	5290	R: GTGGAAGAGTTTGCCTGTTTT	152
qRT-PDK1	100286871	F: AAATCACCAGGACAGCCAATA	190
qRT-PDK1	100286871	R: CTTCTCGGTCACTCATCTTCAC	190
qRT-AKT1	100126861	F: CTGCACAAACGAGGCGAGT	89
qRT-AKT1	100126861	R: CGCTCCTTGTAGCCGATGAA
qRT-FOXO3	733621	F: ACAAACGGCTCACTCTGTCCCA	85
qRT-FOXO3	733621	R: GAACTGTTGCTGTCGCCCTTATC	85
qRT-TSC2	100505408	F: CGAGGTGGTGTCCTACGAGAT	115
qRT-TSC2	100505408	R: GAGCAGGCGTTCAATGATGTT	115
qRT-BAD	100521065	F: AGTCGCCACTGCTCTTACCC	172
qRT-BAD	100521065	R: TCTTGAAGGAACCCTGGAAATC	172
qRT-ESR1	10013276	F: GATGCCTTGGTCTGGGTGAT	124
qRT-ESR1	10013276	R: AGTGTTCCGTGCCCTTGTTA	124
qRT-ESR2	396697	F: AAGGGAAAAGGAGGATGGGACA	202
qRT-ESR2	396697	R: CAGATAGGGACTGCGTGGAGGT	202
qRT-Star	396597	F: GGAAAAGACACAGTCATCACCCAT	121
qRT-Star	396597	R: CAGCAAGCACACACACGGAAC	121
qRT-CYP17	100157047	F: AAGCCAAGACGAACGCAGAAAG	228
qRT-CYP17	100157047	R: TAGATGGGGCACGATTGAAACC	228
qRT-3BHSD	445539	F: GGGGCTTCTGTCTTGATTCCA	284
qRT-3BHSD	445539	R: GGTTTTCAGTGCTTCCTTGTGC	284
qRT-17BHSD	397574	F: CCCAACGCAGGAGACTCAAAAT	149
qRT-17BHSD	397574	R: CCAGAGCCCATAACGAAGACAGA	149
qRT-CYP19A	403332	F: GCTGGACACCTCTAACAACCTCTT	91
qRT-CYP19A	403332	R: TTGCCATTCATCAAAATAACCCT	91

干扰KISS1基因对猪卵巢颗粒细胞功能的影响

Effect of KISS1 Interference on the Function of Porcine Granulosa Cells in Porcine Ovary

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