Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (3): 597-612.doi: 10.3864/j.issn.0578-1752.2024.03.013

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles     Next Articles

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. 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 Online:2024-02-01 Published:2024-02-05

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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

Table 1

Vector sequence information"

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

Table 2

Primer information for RT-qPCR"

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

Fig. 1

Identification and expression level analysis of circZNF423 A. Sanger sequencing; B. RNase R experiments; C. Nucleoplasm separation experiments; D. RNA fluorescence in situ hybridization experiments; E. Immunofluorescence staining assay. *P<0.05, **P<0.01, *** P<0.001; ns, not significant"

Fig. 2

Expression analysis of circZNF423, oar-miR-541-3p and CALM3 in sheep muscle tissue A. Relative expression of circZNF423; B. Relative expression of CALM3; C. Relative expression of oar-miR-541-3p; D Protein expression of CALM3 in sheep muscle tissues with GAPDH as an internal control and Western blot to quantify the results. E. RNAhybrid prediction analysis; F. Dual luciferase reporter gene assay; G. RNA pull-down experiments with biotin-labeled oar-miR-541-3p.*. P<0.05; **. P<0.01; ***. P<0.001; ns, not significant"

Fig. 3

Effect of circZNF423 on the proliferation of sheep myoblasts A. The relative expression of CALM3, Pax7, PCNA, CDK2 and oar-miR-541-3p in sheep myoblasts after overexpression of circZNF423; B. The relative expression of CALM3, Pax7, PCNA, CDK2 and oar-miR-541-3p in sheep myoblasts after inhibition of circZNF423; C. The protein expression of CALM3, Pax7, PCNA and CDK2 in sheep myoblasts after overexpression/inhibition of circZNF423 and the gray value analysis; D, E. The CCK-8 assay of the myoblast proliferation after overexpression or inhibition of circZNF423; F, G. The EdU assay of the myoblast proliferation after overexpression or inhibition of circZNF423. *. P<0.05; **. P<0.01; ***. P<0.001; ns, not significant"

Fig. 4

Effect of oar-miR-541-3p on the proliferation of sheep myogenic cells by regulating the expression of CALM3 and circZNF423 A. The relative expression of CALM3, circZNF423, PCNA, Pax7 and CDK2 after oar-miR-541-3p overexpression; B. The relative expression of CALM3, circZNF423, PCNA, Pax7 and CDK2 after oar-miR-541-3p inhibition. C. The protein expression of CALM3, PCNA, Pax7 and CDK2 after oar-miR-541-3p overexpression or inhibition and the gray value analysis; D, E. The EdU assay of the myoblast proliferation after oar-miR-541-3p overexpression or inhibition; F,G. The CCK-8 assay of the myoblast proliferation after oar-miR-541-3p overexpression or inhibition. *. P<0.05; **. P<0.01; ***P<0.001; ns, not significant"

Fig. 5

Effect of CALM3 on the proliferation of sheep myoblasts A. The relative expression of CALM3, Pax7, PCNA and CDK2 in sheep myoblasts after overexpression of CALM3; B. The relative expression of CALM3, Pax7, PCNA and CDK2 in sheep myoblasts after inhibition of CALM3; C. The protein expression of CALM3, Pax7, PCNA and CDK2 in sheep myoblasts after overexpression/inhibition of CALM3 and the gray value analysis; D, E. The CCK-8 assay of the myoblast proliferation after overexpression or inhibition of CALM3; F, G. The EdU assay of the myoblast proliferation after overexpression or inhibition of CALM3. *. P<0.05; **. P<0.01; ***. P<0.001; ns, not significant"

Fig. 6

Effect of estradiol on the proliferation of sheep myoblasts A. In vitro addition of different concentrations of estradiol was tested for the optimal concentration affecting sheep myogenic cell proliferation; B. Protein expression and gray value analysis of PCNA, Pax7 and CDK2 in sheep myogenic cells after the addition of 10 nmol·L-1 estradiol; C. CCK-8 assay for myoblast proliferation; D. EdU assay for myoblast proliferation; E. Relative expression of circZNF423, oar-miR-541-3p, and CALM3 in sheep myocytes after the addition of 10 nmol·L-1 estradiol; F. Analysis of the protein expression and grayscale values of CALM3 after the addition of 10 nmol·L-1 estradiol. *. P<0.05; **. P<0.01; ns, not significant"

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