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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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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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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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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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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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In this study, the expression of LncMEG3 (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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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. |
| Evidence Sentence: |
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. |
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This result suggests that miR-21-3p is a target of MEG3 (Fig. |
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FSH and E2 production was increased and T, AMH, and LH production was decreased in the sh-MEG3 group compared to the sh-NC group (all P < 0.05) (Fig. |
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Silencing MEG3 increases the number of granulosa cells |
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Silencing of MEG3 attenuated sex hormone dysregulation and ovarian histopathological changes in PCOS rats and promoted follicle cell development and maturation. |
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In addition, silencing MEG3 increased the viability and number of granulosa cells. |
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In addition, silencing MEG3 further inhibited early and late apoptosis of ovarian granulosa cells in PCOS rats. |
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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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The expression of MEG3 (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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Silencing of MEG3 ameliorates abnormal sex hormone expression and ovarian histopathological changes in PCOS rats and promotes follicle cell development and maturation |
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Sh-MEG3 rats showed diffuse cystic mild to moderate dilatation of the ovaries with a few corpus luteum and atretic follicles (Fig. |
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The numbers of mature follicles and corpus luteum structures were increased in the sh-MEG3 group compared to the sh-NC group (P < 0.01) (Fig. |
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After MEG3 silencing lentivirus intervention in PCOS rats, granulosa cells extracted from follicles were cultured in an incubator at 37 C for 24 h. The growth of ovarian granulosa cells were observed after they adhered to the well. |
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There were more granulosa cells in the sh-MEG3 group than in the sh-NC and PCOS groups (P < 0.0001) (Fig. |
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Silencing of MEG3 inhibits the apoptosis of ovarian granulosa cells in PCOS rats |
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After MEG3 silencing lentivirus intervention in PCOS rats, an increase in mitochondrial membrane potential (JC-1) could be detected in granulosa cells, so that the green fluorescence changed to red fluorescence (Fig. |
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Compared with the sh-NC and PCOS groups, the sh-MEG3 group showed decreased caspase-3 mRNA expression and decreased cleaved caspase-3/caspase-3 ratio, and increased Bcl-2mRNA expression, decreased Bax mRNA expression and increased Bcl-2/Bax ratio (all P < 0.01) (Fig. |
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The results indicated that silencing MEG3 repaired the mitochondrial membrane potential and associated apoptotic proteins in the early stages of granulosa cell apoptosis. |
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The fluorescence values of ovarian granulosa cells in the sh-MEG3 group were lower than those in the sh-NC group (P < 0.01), and a few apoptotic cells showed green fluorescence (Fig. |
| Evidence Sentence: |
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. |
| Evidence Sentence: |
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. |
