miR-21-3p inhibits autophagy of bovine granulosa cells by targeting VEGFA via PI3K/AKT signaling

in Reproduction
Correspondence should be addressed to Z Jiang; Email: jiangzhongliang@nwafu.edu.cn

*(L Ma and Y Zheng contributed equally to this work)

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It is well documented that granulosa cell apoptosis is the main reason for follicular atresia and death; however, increasing evidence suggests that autophagy plays an important role in the fate of granulosa cells. miR-21-3p regulates many fundamental biological processes and is pivotal in the autophagy of tumor cells; nevertheless, the autophagy in cattle ovary and how miR-21-3p regulates the follicular cells is unknown. In this study, we aimed to elucidate the autophagy and the role of miR-21-3p in cattle ovary using bovine primary ovarian granulosa cells (BGCs). The results showed the autophagy for the first time in BGCs in large follicle according to autophagic gene transcript of LC3, BECN-1, ATG3, protein expression of LC3, P62 and LC3 puncta, a standard marker for autophagosomes. miR-21-3p was identified as a novel miRNA that repressed BGCs autophagy according to the results from plasmids transfection of miR-21-3p mimics and inhibitor. Meanwhile, VEGFA was confirmed to be a validated target of miR-21-3p in BGCs using luciferase reporter assays and the results of VEGFA expression decreased with transfection of miR-21-3p mimics, while it increased with transfection of miR-21-3p inhibitor. In addition, small interference-mediated knockdown of VEGFA significantly inhibits BGCs autophagy signaling; however, overexpression of VEGFA in BGCs promoted autophagy in the presence of miR-21-3p. Finally, the results of AKT and its phosphorylation suggested that miR-21-3p suppressed VEGFA expression through downregulating AKT phosphorylation signaling. In summary, this study demonstrates that miR-21-3p inhibits BGCs autophagy by targeting VEGFA and attenuating PI3K/AKT signaling.

 

    Society for Reproduction and Fertility

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    Expression of autophagic marker genes and miR-21-3p in BGCs. (A) Cells were aspirated from follicles using a syringe, and real-time PCR analysis of autophagic marker genes and miR-21-3p expression in BGCs from small, medium and large follicles. (B) Western blot analysis of LC3 levels in the BGCs of follicles. (C) GFP-LC3 puncta accumulate in the follicles. BGCs were transfected with GFP-LC3 plasmids, and autophagy was assessed after 24 h. (D) BCL-2 and BAX mRNA levels and the ratio of BCL-2/BAX in BGCs were determined. Bar = 20 μm. Data are means ± s.e.m. of three independent experiments, bars with no common asterisk are significantly different (P < 0.05). Note: small: small follicle; medium: medium follicle; large: large follicle.

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    miR-21-3p inhibits BGC autophagy. (A) Cells were transfected with miR-21-3p mimic or inhibitor, and after 24 h, autophagy was assessed by MDC staining. Bars = 20 μm. Percentage of MDC-positive cells (the puncta) were calculated and represented in the graph. (B) Real-time PCR analysis of LC3, BECN-1, ATG3 and miR-21-3p after transfection with miR-21-3p mimic to cells for 6 h and cultured for 42 h with fresh medium. (C) Real-time PCR analysis of LC3, BECN-1, ATG3 and miR-21-3p after transfection with miR-21-3p inhibitor to cells for 6 h and cultured for 42 h with fresh medium. (D) Western blot analysis of LC3 and P62 after transfection miR-21-3p mimic or inhibitor to cells for 6 h and cultured for 42 h with fresh medium. Band intensities were calculated using Image J software. Data are means ± s.e.m. of three independent replicate, bars with no common asterisk are significantly different (P < 0.05).

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    miR-21-3p targets VEGFA. (A) Binding sites of miR-21-3p and VEGFA. (B) Luciferase reporter assays showing that VEGFA is a direct target of miR-21-3p. (C) Real-time PCR analysis showing effects of miR-21-3p mimic and inhibitor on VEGFA mRNA levels in BGCs after treatment for 6 h and cultured for 42 h with fresh medium. (D) Western bolt analysis showing effects of miR-21-3p on VEGFA expression after plasmids transfection for 6 h, and cultured for 42 h with fresh medium. Data are means ± s.e.m. of three independent replicates, bars with no common asterisk are significantly different (P < 0.05).

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    Knockdown of VEGFA inhibits autophagy in BGCs. (A) Real-time PCR analysis for LC3, BECN-1, ATG3 and VEGFA mRNA after transfection with si-Vegfa for 6 h and cultured for 42 h with fresh medium. (B) Western blot analysis of LC3, VEGFA and P62 expression after transfection with si-Vegfa for 6 h and cultured for 42 h with fresh medium. (C) Western blot analysis of LC3, VEGFA and P62 after co-transfection with miR-21-3p inhibitors and si-VEGFA for 6 h and cultured for 42 h with fresh medium. Data are means ± s.e.m. of three independent replicate, bars with no common asterisk are significantly different (P < 0.05).

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    Overexpression of VEGFA promotes autophagy in BGCs. (A) Real-time PCR analysis of LC3, BECN-1, ATG3 and VEGFA after transfection with oe-Vegfa for 6 h and cultured for 42 h with fresh medium. (B) Western blot analysis of LC3, VEGFA and P62 in the presence of oe-VEGFA treatment for 6 h and cultured for 42 h with fresh medium. (C) Western blot analysis of LC3, VEGFA and P62 after co-transfection of the miR-21-3p inhibitor and oe-Vegfa for 6 h and cultured for 42 h with fresh medium. Data are means ± s.e.m. of three independent replicate, bars with no common asterisk are significantly different (P < 0.05).

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    miR-21-3p downregulates AKT phosphorylation in BGCs. Western blot analysis of AKT and p-AKT in BGCs following transfected with (A) miR-21-3p mimics or inhibitor; (B) si-Vegfa; and (C) oe-Vegfa plasmids for 6 h and cultured for 42 h with fresh medium. (D) A model of how miR-21-3p regulates autophagy in BGCs. miR-21-3p directly targets VEGFA, resulting in the downregulation of p-AKT/AKT signaling, promoting BGC autophagy. ‘—’ means the inhibition function. Data are means ± s.e.m. of three independent replicate, bars with no common asterisk are significantly different (P < 0.05).

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