关键词: 多囊卵巢综合征, 卵巢颗粒细胞, 凋亡, miR-144-3p, PTEN

Abstract: Objective To investigate the expressions of miR-144-3p and its target gene PTEN in ovarian granule cells and their functions in patients with polycystic ovary syndrome. Methods Twenty patients with PCOS and 20 controls were recruited. Granulosa cells were collected in both groups and the expression level of miR-144-3p in cells was analyzed. The target genes of miR-144-3p were predicted by TargetScan database, and the binding relationship was verified by dual luciferase gene reporter assay. The human ovarian granulosa cells ( KGN) were resuscitated and transfected with miR-144-3p mimic / mimic NC, or miR-144-3p inhibitor / inhibitor NC, or si-PTEN / siRNANC, and were then accordingly divided into 6 groups: miR-144-3p mimic group, mimic NC group, miR-144-3p inhibitor group, inhibitor NC group, si-PTEN and siRNA-NC group. Cell apoptosis was detected by TUNEL assay. RT-PCR and Western blotting method were used to detect the relative expression levels of miR-144-3p and PTEN. Results RT-PCR results showed that the expression level of miR-144-3p in the PCOS group was significantly lower than that in the control group(P< 0. 05). Flow cytometry results showed that the apoptosis rate of granulosa cells in the miR-144- 3p mimic group was significantly lower than that in the mimic-NC group (P< 0. 05), while the apoptosis rate of granulosa cells in the miR-144-3p inhibitor group was significantly higher than that inthe inhibitor-NC group (P< 0. 05). PTEN was predicted as the target gene of miR-144-3p by bioinformatics analysis, and the luciferase gene reporter experiments had confirmed that miR-144-3pcould specifically bind to the PTEN-3 ′ UTR and down-regulate the expression level of PTEN gene. RT-PCR assay results showed that the expression level of PTEN mRNA in the PCOS group was significantly higher than that in the control group (P < 0. 05), and was negatively correlated with the expression level of miR-144-3p (r = - 0. 91, P< 0. 001). The results of qRT-PCR and Westernblotting showed that the mRNA and protein expression levels of PTEN in the miR-144-3p mimicgroup were significantly lower than those in the NC mimic group (P< 0. 05), while the mRNA andprotein expression levels of PTEN in the miR-144-3p inhibitor group were significantly higher thanthose in the inhibitor NC group (P< 0. 05). Flow cytometry showed that the apoptosis rate of granulosa cells in the si-PTEN group was significantly lower than that in the siRNA-NC group ( P <0. 05), while the apoptosis rate of granulosa cells in the miR-144-3p inhibitor + si-PTEN group wassignificantly lower than that in the miR-144-3p inhibitor group (P < 0. 05). Conclusion The expression level of miR-144-3p in ovarian granule cells in PCOS patients is significantly reduced,while the expression level of PTEN gene is significantly increased, which may have promoted theapoptosis of ovarian granule cells in PCOS patients.

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