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Acupuncture regulates the apoptosis of ovarian granulosa cells in polycystic ovarian syndrome-related abnormal follicular development through LncMEG3-mediated inhibition of miR-21-3p |
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This study hypothesized that the mechanism of acupuncture on follicular development abnormalities in PCOS patients is the inhibition of granulosa cell apoptosis through LncMEG3-mediated regulation of miR-21-3p. |
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Primary granulosa cells were isolated from each group of rats to assess the association of acupuncture treatment, LncMEG3, miR-21-3p, and granulosa cell apoptosis in rats with PCOS. |
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LncMEG3 and miR-21-3p were highly expressed in the ovarian granulosa cells of rats with PCOS, and LncMEG3-mediated regulation of miR-21-3p was involved in the development of PCOS in rats. |
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Acupuncture intervention inhibited early and late apoptosis of ovarian granulosa cells in PCOS rats by targeting miR-21-3p via LncMEG3. |
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These results suggest that acupuncture can downregulate LncMEG3, thereby targeting and regulating miR-21-3p to suppress early and late granulosa cell apoptosis and normalize their proliferation. |
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LncMEG3-mediated inhibition of miR-21-3p regulates ovarian granulosa cell apoptosis. |
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LncMEG3 and miR-21-3p are involved in the occurrence and development of PCOS-related abnormal follicular development. |
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High expression of LncMEG3 and miR-21-3p in ovarian granulosa cells found in rats with PCOS |
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LncMEG3 is involved in the progression of human diseases by regulating miR-21-3p, but its role in PCOS is unclear. |
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1A) and miR-21-3p (Fig. |
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1B) in ovarian granulosa cells of PCOS rats was detected by RT-qPCR and Western blot analysis, and the results showed that the expression of LncMEG3 and miR-21-3p was increased in the PCOS group compared with the Normal group (both P < 0.0.001). |
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To investigate the correlation of the LncMEG3/miR-21-3p axis in PCOS, we applied the Pearson test to analyse the relationship between LncMEG3 and miR-21-3p expression, and the results showed (Fig. |
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1C) a positive correlation between LncMEG3 expression and miR-21-3p expression in ovarian granulosa cells of PCOS patients (r = 0.9831, P < 0.0001). |
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LncMEG3 targets and regulates miR-21-3p |
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To further analyse the relationship between LncMEG3(RGD1566401) and miR-21-3p and the relationship between LncMEG3 and miR-21-3p in PCOS, the binding sites between LncMEG3 and miR-21-3p were analysed, and pmirGLO-RGD1566401-WT and pmirGLO-RGD1566401-Mut plasmids were constructed (Fig. |
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Dual-luciferase reporter gene analysis showed that rno-miR-21-3p significantly decreased the luciferase activity of the r-RGD1566401-WT construct compared with that of the NC construct (P < 0.0001), indicating a binding interaction between the two sites; after mutation, rno-miR-21-3p failed to decrease r RGD1566401-MUT luciferase activity (P > 0.05), indicating that the mutation was successful. |
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This result suggests that miR-21-3p is a target of MEG3 (Fig. |
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To investigate the specific mechanism by which the MEG3/miR-21-3p axis is involved in the development of follicular abnormalities in PCOS, we used a rat model of PCOS induced by DHEA, along with silent MEG3 lentivirus intervention in PCOS rats (Figs. |
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2D) and miR-21-3p (Fig. |
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The results showed that the expression of MEG3 and miR-21-3p was enhanced in the ovarian granulosa cells of the DHEA group compared with those of the normal group (P < 0.0001 for both). |
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The expression of MEG3 and miR-21-3p was reduced in the sh-MEG3 group compared with the sh-NC group (both P < 0.0001). |
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Inhibition of apoptosis of ovarian granulosa cells in PCOS rats through LncMEG3-mediated regulation of miR-21-3p after acupuncture intervention |
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To further investigate the mechanism of action by which acupuncture inhibits the apoptosis of ovarian granulosa cells in rats with PCOS, we extracted primary ovarian granulosa cells from rats with PCOS after acupuncture intervention and detected the transcription of granulosa cell LncMEG3 and miR-21-3p, apoptotic proteins (including cleaved caspase-3, Bax and Bcl-2), mitochondrial membrane potential (JC-1), and TUNEL staining to detect early, mid and late-stage apoptosis. |
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The transcription of LncMEG3, miR-21-3p, caspase-3, Bax and Bcl-2 was detected by RT-qPCR, and the apoptosis of ovarian granulosa cells was detected by Western blot, and mitochondrial membrane potential (JC-1), and TUNEL assays. |
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8A), the PCOS group showed increased caspase-3, LncMEG3 and miR-21-3p mRNA transcription, increased cleaved caspase-3/caspase-3 ratio, decreased Bcl-2 mRNA transcription, increased Bax mRNA transcription and decreased Bcl-2/Bax ratio compared to the normal group (all P < 0.0001) (Fig. |
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8L, M), indicating that acupuncture regulates miR-21-3p via LncMEG3 to inhibit ovarian granulosa cell apoptosis in PCOS rats. |
