Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (23): 4757-4771.doi: 10.3864/j.issn.0578-1752.2023.23.016

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles     Next Articles

miR-535 Targets the GAB2 Gene to Promote Goat Granulosa Cell Proliferation Through Activation of the PI3K/AKT Signaling Pathway

WANG Peng(), LIU ZiYi, LIU YuFang(), CHU MingXing()   

  1. Institute of Animal Science, Chinese Academy of Agricultural Sciences/State Key Laboratory of Animal Biotech Breeding, Beijing 100193
  • Received:2023-03-24 Accepted:2023-08-31 Online:2023-12-04 Published:2023-12-04
  • Contact: LIU YuFang, CHU MingXing

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Abstract:

【Background】MicroRNAs (miRNAs) are short RNA molecules of 18-25 nt in length that play an important role in the regulation of follicle development in mammalian ovary granulosa cells (GCs). The previous sequencing of the transcriptome of the ovaries of high and low kidding individuals in Yunshang black goats showed that miR-535 was able to influence the kidding number of goats, but the specific regulatory mechanism was not yet clear. 【Objective】 The aim of this study was to investigate the molecular mechanisms of miR-535 targeting the GRB2 associated binding protein 2 (GAB2) and its associated signaling pathway PI3K/AKT affected the proliferation of goat GCs, so as to further investigate the molecular biological regulation mechanism. 【Method】In this study, three high- and low-fertility Yunshang black goats with the kidding number record of more than two litters were selected, and their follicular ovarian tissues were collected after synchronous estrus treatment for collecting primary GCs. The expression of miR-535 and GAB2 in high- and low-yield ovary tissues of Yunshang black goats was detected by reverse transcription-quantitative PCR (RT-qPCR). The overexpression/inhibitor of GAB2 vector was constructed and the effect of candidate GAB2 on the proliferation of goat GCs was detected using RT-qPCR, Western blot, immunofluorescence, CCK8, EdU and Apoptosis, respectively. The prediction of the targeted relationship between miR-535 and GAB2 was performed with miRDB and miRanda software. The Wild-type and Mutant vectors of GAB2 were constructed and the targeting relationship between miR-535 and GAB2 was detected by the dual luciferase activity assay. The overexpression/inhibitor miR-535 vector was constructed to explore the effect of its GCs proliferation and downstream gene function. 【Result】 The RT-qPCR results showed that the expression of GAB2 was significantly lower in ovarian tissues of Yunshang black goats with high-fertility than that in low-fertility groups, and the expression of miR-535 was the opposite (P<0.05). The results of RT-qPCR and Western blot showed that the expression of CCND2, CDK4 and BCL2 was significantly increased (P<0.05), while the expression of BAX was significantly decreased (P<0.05) after overexpression of GAB2 in GCs, and the inhibition of their expression was the opposite. Both EdU and CCK8 assays showed that GAB2 overexpression significantly promoted the proliferation of granulosa cell, while inhibition of its expression was the opposite (P<0.05). Dual luciferase reporter assays showed that miR-535 inhibited dual luciferase activity in the 3'UTR region of the GAB2 gene. The results of RT-qPCR and Western blot showed that the expression of GAB2, CCND2, CDK4 and BCL2 in goat GCs was significantly decreased and the expression of BAX was significantly increased after miR-535 overexpression, while the opposite was true after miR-535 inhibition (P<0.05). Both EdU and CCK8 assays showed that miR-535 overexpression significantly inhibited the proliferation of GCs, while the opposite was true after miR-535 inhibition (P<0.05). Apoptosis assays showed that miR-535 overexpression promoted GCs proliferation and the opposite was true after inhibition of its expression. The expression levels of the PI3K/AKT signaling pathway marker AKT in goat GCs were significantly increased after inhibition of miR-535, respectively (P<0.05). 【Conclusion】 In conclusion, the results of this study suggested that miR-535 inhibited the proliferation of goat granulosa cells by suppressing the expression of GAB2. These results provided a theoretical basis for further investigation of the biological functions of miR-535 regulating goat GCs.

Key words: goat kidding number, ovarian granulosa cell proliferation, GAB2 gene, miR-535, P13K/AKT signaling pathway

Table 1

Primer sequences used for RT-qPCR"

基因名称
Gene name		 引物序列
Primer sequence (5′→ 3′)		 Genbank
Cyclin-D2 F: CCTTTCGCTTACCTATACC XM_005680985.3
R: ATGTGGATTGCCTCAAAGCC
CDK4 F: CAGGTCGATATCCCGAACATC
R: GAGCATCCCAATGTTGTCAGG XM_005680266.3
BAX F: ACTGGCGCATCAGATCCTTT
R: CCAAGAAGCTGAGCGAGTGTCTG XM_013971446.2
BCL2 F: GTGTCCACGGCTGCGATCATC
R: TGTGGATGACCGAGTACCTGAACC NM_001314213.1
GAB2 F: GCCAGACTGAGCAGTGCCTTC
R: CTATGCCTGGAAGAAACGC XM_018043071.1
miR-535 F: CTCGCTGGTCTTGATGTCG
R: ATCCAGTGCAGGGTCCGAGG -
U6 CAAGGATGACACGCAAATTCG -

Fig. 1

Expression of miR-535 and GAB2 in ovarian tissues of Yunshang black goats with high and low-fertility A. The expression of miR-535 in Yunshang black goat ovarian tissues with high- and low-fertility; B. The expression of GAB2 in Yunshang black goat ovarian tissues with high- and low-fertility. *P<0.05; **P<0.01. The same as below"

Fig. 2

GAB2 promoted the proliferation of goat granulosa cells and inhibited their apoptosis A. Immunofluorescence detection of GAB2 expression after overexpression or inhibition of GAB2; B. Relative mRNA expression levels of CCND2, CDK4, BCL2 and BAX after GAB2 overexpression or inhibition; C and D. The protein expression levels and gray value analysis of CCND2, CDK4, BCL2 and BAX after overexpression or inhibition of GAB2; E. EdU assay of granulosa cell proliferation after overexpression or inhibition of GAB2; F. CCK8 assay of granulosa cell proliferation after overexpression or inhibition of GAB2; G. Apoptosis detection of granulosa cell proliferation after overexpression or inhibition of GAB2"

Fig. 3

Targeted regulation of GAB2 expression by miR-535 in goat granulosa cells A. Prediction of miR-535 and GAB2 binding sites conservatism; B. Dual luciferase activity assay of miR-535 mimics and GAB2 3'UTR region; C. The mRNA level expression of GAB2 after miR-535 overexpression or inhibition; D, E. Detection of GAB2 protein level after miR-535 overexpression or inhibition and the gray value analysis"

Fig. 4

miR-535 inhibited proliferation and promoted apoptosis in goat granulosa cells A. Relative mRNA expression of miR-535 after transfection with miR-535 mimics, miR-535 inhibitor and NC;B. Relative mRNA expression levels of CCND2, CDK4, BCL2 and BAX after miR-535 overexpression; C. Relative mRNA expression levels of CCND2, CDK4, BCL2 and BAX after miR-535 inhibition; D and E. The protein expression levels and grayscale values of CCND2, CDK4, BCL2 and BAX after overexpression or inhibition of miR-535; F. Detection of granulosa cell proliferation by EdU after overexpression or inhibition of miR-535; G. Detection of granulosa cell proliferation by CCK8 after overexpression or inhibition of miR-535 granulosa cell proliferation; H. Apoptosis detection of granulosa cells after overexpression or inhibition of miR-535"

Fig. 5

Antagonizes effect of GAB2 on regulation of miR-535"

Fig. 6

miR-535 targeted to regulate GAB2 to activate PI3K/AKT signaling pathway A. STRING analysis of GAB2 related proteins; B. Relative mRNA expression of AKT1 after overexpression or inhibition of miR-535; C. The protein expression of AKT1 after overexpression or inhibition of miR-535; D. The gray value analysis of protein expression levels of AKT1 after overexpression or inhibition of miR-535"

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