雌激素介导circZNF423作为ceRNA调控oar-miR-541-3p/CALM3通路促进绵羊成肌细胞增殖

doi:10.3864/j.issn.0578-1752.2024.03.013

中国农业科学 ›› 2024, Vol. 57 ›› Issue (3): 597-612.doi: 10.3864/j.issn.0578-1752.2024.03.013

雌激素介导circZNF423作为ceRNA调控oar-miR-541-3p/CALM3通路促进绵羊成肌细胞增殖

池润清¹^,²(), 韩海银¹(), 王鹏¹, 李凯扬³, 储明星²(), 刘玉芳²()

¹ 河北工程大学生命科学与食品工程学院，河北邯郸 056021
² 中国农业科学院北京畜牧兽医研究所/农业农村部动物遗传育种与繁殖重点实验室，北京 100193
³ 北京市畜牧总站，北京 100107

收稿日期:2023-06-07 接受日期:2023-09-10 出版日期:2024-02-01 发布日期:2024-02-05
通信作者:
刘玉芳，E-mail：aigaiy@126.com
储明星，E-mail：mxchu@263.net
联系方式: 池润清，E-mail：1406049336@qq.com。韩海银，E-mail：1299788389@qq.com。池润清与韩海银为同等贡献作者。
基金资助:
国家现代农业产业技术体系(CARS-38); 中国农业科学院科技创新工程(CAAS-ZDRW202106); 中国农业科学院科技创新工程(ASTIP-IAS13)

Estrogen Mediates CircZNF423 as a Sponge for oar-miR-541-3p to Target CALM3 for Regulating Myoblast Proliferation in Sheep

CHI RunQing¹^,²(), HAN HaiYin¹(), WANG Peng¹, LI KaiYang³, CHU MingXing²(), LIU YuFang²()

¹ College of Life Science and Food Engineering, Hebei University of Engineering, Handan 056021, Hebei
² Key Laboratory of Animal Genetics, Breeding and Reproduction of Ministry of Agriculture and Rural Affairs/Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193
³ Beijing General Station of Animal Husbandry, Beijing 100107

Received:2023-06-07 Accepted:2023-09-10 Published:2024-02-01 Online:2024-02-05

摘要/Abstract

摘要：

【背景】雌激素是雌性哺乳动物卵巢组织分泌的主要激素，其对肌肉的生长发育起着重要的调控作用，circRNA已被发现参与多种与肌肉生长发育相关的信号通路。【目的】根据团队前期卵巢摘除苏尼特羊与假手术苏尼特羊全转录组整合分析发现circZNF423可作为内源性竞争RNA（ceRNA）调控oar-miR-541-3p/CALM3的表达。为进一步探究雌激素在绵羊肌肉生长中分子机制，通过体外培养绵羊原代成肌细胞，检测成肌细胞中外源添加雌激素介导circZNF423调控oar-miR-541-3p/CALM3对成肌细胞增殖的影响，为进一步研究雌激素及circRNA在绵羊生长发育性状中的作用机制提供理论依据，为绵羊分子设计育种提供新的研究思路。【方法】采集绵羊背最长肌组织，对绵羊原代成肌细胞进行体外分离培养，通过免疫荧光染色、RNA原位杂交（FISH）和核质分离试验确定circZNF423在成肌细胞中的表达位置；RNAhybrid在线软件预测circZNF423、oar-miR-541-3p和CALM3存在结合关系，通过双荧光素酶活性检测和生物素标记的miRNA下拉试验验证circZNF423和oar-miR-541-3p、oar-miR-541-3p和CALM3结合情况；体外构建合成circZNF423过表达或干扰载体、oar-miR-541-3p的模拟物（mimics）或抑制剂（inhibitor）、CALM3过表达或干扰载体，在绵羊原代成肌细胞中进行转染，利用RT-qPCR、Western blot、EdU和CCK-8检测绵羊成肌细胞增殖标志因子的表达和成肌细胞增殖情况；为进一步明确雌激素在绵羊肌肉生长发育中的作用，体外添加不同浓度的外源性雌二醇（E₂），利用RT-qPCR、Western blot、CCK-8和EdU检测绵羊肌细胞增殖标志基因的表达变化。【结果】免疫荧光染色显示分离的原代细胞为绵羊成肌细胞，可用于后续功能验证；RNA原位杂交和核质分离试验表明，circZNF423主要在绵羊成肌细胞质中表达。RNAhybrid、双荧光素酶活性检测和生物素标记的miRNA下拉试验结果表明，circZNF423与oar-miR-541-3p、oar-miR-541-3p与CALM3 3’UTR之间均存在显著的结合关系；抑制circZNF423/CALM3表达或过表达oar-miR-541-3p后，绵羊成肌细胞增殖标志因子 PCNA、CDK2和Pax7 mRNA与蛋白表达水平一致，均显著上调（P<0.05或P<0.01）；EdU和CCK8结果表明，抑制circZNF423/CALM3表达或过表达oar-miR-541-3p后，绵羊成肌细胞增殖率显著上升（P<0.05）；过表达circZNF423/CALM3或抑制oar-miR-541-3p后则相反。添加不同浓度外源雌二醇（E₂）后，在浓度为10 nmol·L^-1时绵羊成肌细胞增殖标志因子表达最高，显著高于1和100 nmol·L^-1的添加浓度（P<0.05或P<0.01）；绵羊肌细胞增殖标志因子 PCNA、CDK2和Pax7 mRNA和蛋白表达水平一致，均显著上调，circZNF423和CALM3的表达量显著降低，oar-miR-541-3p的表达量显著升高（P<0.05或P<0.01）；EdU和CCK8结果显示，体外添加雌二醇后绵羊成肌细胞增殖率显著升高（P<0.05）。【结论】绵羊成肌细胞中circZNF423作为ceRNA调控oar-miR-541-3p和CALM3的结合及表达，添加外源雌激素可通过抑制circZNF423/oar-miR-541-3p/CALM3通路促进绵羊肌肉细胞的增殖。这些结果为揭示雌激素及circZNF423在绵羊骨骼肌发育性状中的分子机制提供理论基础。

关键词: 绵羊, 肌肉发育, 雌激素, circZNF423, oar-miR-541-3p, CALM3

Abstract:

【Background】Estrogen is the main hormone secreted by female mammalian ovarian tissue, and it plays an important regulatory role in muscle growth and development. The circRNA has been found to be involved in a variety of signaling pathways related to muscle growth and development. 【Objective】According to the whole transcriptome integration analysis of ovariectomized and not ovariectomized Sunite sheep in our previous study, the circZNF423 was found to regulate the expression of oar-miR-541-3p/CALM3 as ceRNA. To further investigate the molecular mechanism of estrogen in sheep muscle growth, the effect of exogenous addition of estrogen-mediated regulation of oar-miR-541-3p/CALM3 by circZNF423 on myoblast proliferation was examined by sheep primary myoblast in vitro culture. This study could provide a theoretical basis for further study of the role of estrogen and circRNA in sheep growth and development trait, and also provide new research ideas for sheep molecular design breeding. 【Method】In this study, the sheep longissimus dorsi tissue was collected, and the sheep primary myoblast were isolated and cultured in vitro. The location of circZNF423 expression in myoblast was determined by immunofluorescence staining, RNA in situ hybridization (FISH) and nucleoplasmic separation experiments. RNA hybrid online software was used to predict the existence of binding relationship between circZNF423, oar-miR-541-3p and CALM3, and the binding of circZNF423 to oar-miR-541-3p, oar-miR-541-3p and CALM3 was verified by dual luciferase activity assay and biotin-labeled miRNA pull-down assay. The synthetic circZNF423 overexpression or interference plasmids, oar-miR-541-3p mimics (mimics) or inhibitors (inhibitors), and CALM3 overexpression or interference vectors were constructed in vitro and transfected in primary sheep myoblast, and the expression of proliferation markers and myoblast proliferation rate were detected using RT-qPCR, Western blot, EdU and CCK-8. To further clarify the role of estrogen in sheep muscle growth and development, the exogenous estradiol (E₂) was added at different concentrations in vitro, and the changes in the expression of sheep myoblast proliferation were detected using RT-qPCR, Western blot, CCK-8, and EdU. 【Result】Immunofluorescence staining showed that the isolated primary myoblast were sheep myoblast and could be used for subsequent functional validation experiments. RNA in situ hybridization and nucleoplasmic isolation experiments showed that circZNF423 was mainly expressed in the cytoplasm of sheep myoblast. The results of RNAhybrid, dual luciferase activity assay and biotin-labeled miRNA pull-down assay showed a significant binding relationship between both circZNF423 and oar-miR-541-3p, oar-miR-541-3p and CALM3 3’UTR. The expression of proliferation markers PCNA, CDK2 and Pax7 in sheep myoblast was significant increased after circZNF423/CALM3 inhibition or oar-miR-541-3p overexpression (P<0.05 or P<0.01). The EdU and CCK8 results indicated that the proliferation rate of sheep myoblast was significantly increased after circZNF423/ CALM3 inhibition or oar-miR-541-3p overexpression (P<0.05), while the opposite was true after circZNF423/CALM3 overexpression or oar-miR-541-3p inhibition. The expression of sheep myoblast proliferation markers was significantly higher at 10 nmol·L^-1 than that at 1 nmol·L^-1 and 100 nmol·L^-1 after the addition of different concentrations of exogenous estradiol (E₂) (P<0.05 or P<0.01). The mRNA and protein expression levels of the proliferation markers PCNA, CDK2 and Pax7 significantly increased in sheep myoblast, while the expression of circZNF423 and CALM3 were significantly decreased and the expression of oar-miR-541-3p was significantly increased (P<0.05 or P<0.01). The results of EdU and CCK8 showed that the proliferation rate of sheep myoblast was significantly increased after the addition of estradiol in vitro (P<0.05). 【Conclusion】CircZNF423 as ceRNA regulated the binding and expression of oar-miR-541-3p and CALM3 in sheep myoblast, and the addition of exogenous estrogen promoted the proliferation of sheep myoblast by inhibiting the circZNF423/oar-miR-541-3p/CALM3 pathway. These results provided a theoretical basis for revealing the molecular mechanisms of estrogen and circZNF423 in the developmental traits of sheep skeletal muscle.

Key words: sheep, muscle development, estrogen, circZNF423, oar-miR-541-3p, CALM3

池润清, 韩海银, 王鹏, 李凯扬, 储明星, 刘玉芳. 雌激素介导circZNF423作为ceRNA调控oar-miR-541-3p/CALM3通路促进绵羊成肌细胞增殖[J]. 中国农业科学, 2024, 57(3): 597-612.

CHI RunQing, HAN HaiYin, WANG Peng, LI KaiYang, CHU MingXing, LIU YuFang. Estrogen Mediates CircZNF423 as a Sponge for oar-miR-541-3p to Target CALM3 for Regulating Myoblast Proliferation in Sheep[J]. Scientia Agricultura Sinica, 2024, 57(3): 597-612.

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载体名称 Vector name	载体序列 Vector sequence (5′-3′)
siRNA-circZNF423	F: UCCUGGAGUUGAAGAGGGGTT
siRNA-circZNF423	R: CCCCUCUUCAACUCCAGGATT
siRNA-CALM3	F: GAGGAGGUGGACGAAAUGATT
siRNA-CALM3	R: UCAUUUCGUCCACCUCCUCTT
oar-miR-541-3p mimics	F: UGGUGGGCACAGAAUCCGGCCUCU
oar-miR-541-3p mimics	R: AGGCCGGAUUCUGUGCCCACCAUU
mimics NC/ siRNA NC	F: UUCUCCGAACGUGUCACGUTT
mimics NC/ siRNA NC	R: ACGUGACACGUUCGGAGAATT
oar-miR-541-3p inhibitor	F: AGAGGCCGGAUUCUGUGCCCACCA
inhibitor NC	F: CAGUACUUUUGUGUAGUACAA

基因名称 Gene name	引物序列 Primer sequence (5′-3′)	退火温度 Tm (℃)
circZNF423	F: CTGGAAGACAGGAACAGCGT	60
circZNF423	R: CTGACAGTGATCGCAGGTGT	60
oar-miR-541-3p	F: TGGTGGGCACAGAATCCG	58
oar-miR-541-3p	R: CAGTTTTTTTTTTTTTTTGGGCAG	58
CALM3	F: GGAGCAGATCGCAGAGTTCA	60
CALM3	R: GCTCTGCGGCACTGATGTAC	60
PCNA	F: TTGAAGAAAGTGCTGGAGGC	60
PCNA	R: TTGGACATGCTGGTGAGGTT	60
CDK2	F: AAGTGGCTGCATCACAAGGA	60
CDK2	R: CAAGCTCCGTCCATCTTCAT	60
Pax7	F: CGTGCCCTCAGTGAGTTCGA	60
Pax7	R: CCAGACGGTTCCCTTTGTCG	60
β-actin	F: AGCCTTCCTTCCTGGGCATGGA	60
β-actin	R: GCTCTGCGGCACTGATGTAC	60
U6	F: AACGCTTCACGAATTTGCGT	60
U6	R: CTCGCTTCGGCAGCACA	60
GAPDH	F: CACGGCACAGTCAAGGCAG	60
GAPDH	R: AGATGATGACCCTCTTGGCG	60

雌激素介导circZNF423作为ceRNA调控oar-miR-541-3p/CALM3通路促进绵羊成肌细胞增殖

Estrogen Mediates CircZNF423 as a Sponge for oar-miR-541-3p to Target CALM3 for Regulating Myoblast Proliferation in Sheep

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