pLVX-EGFP-C1

Polycystic ovary syndrome(PCOS)is a common endocrine and metabolic disease.Guizhi Fuling Wan(GZFLW)is commonly used to treat various gynecological diseases.Similar to metformin,GZFLW can effectively improve insulin resistance in PCOS patients,promote mature follicle development and ovulation,and improve insulin sensitivity in insulin-resistant PCOS model rats by reshaping intestinal homeostasis.The study here explores the potential molecular mechanism by which the GZFLW inhibits autophagy in granulosa cells(GCs)in polycystic ovary syndrome(PCOS).GZFLW intervention reduced the expression of autophagy-related proteins PTEN,MMP-2 and Bax by up-regulating the expression of miR-29b-3p and down-regulating the expression of H19.GZFLW treatment significantly reduced the number of autophagosomes and autophagic polymers.However,inhibition of miR-29b-3p and overexpression of H19 induced a significant increase in the number of autophagosomes and autophagic polymers,thereby attenuating the inhibitory effect of GZFLW on autophagy.

In this study,wild-type or mutant H19-MT,PTEN-MT,Bax-MT,and MMP-2-MT luciferase reporter plasmids were constructed using pLVX-EGFP-C1(purchased from Creative Biogene)and pMIR-REPORTE plasmids.Cells were co-transfected with wild-type or mutant luciferase reporter vectors and miR-29b-3p mimic or mimic-NC for 48 h.The results showed that H19 is a direct target of miR-29b-3p,and co-transfection of miR-29b-3p analogues with H19-WT significantly inhibited luciferase activity.Compared with the control group,the expression of H19 in GCs in the PCOS model group was significantly increased,and the expression of miR-29b-3p was significantly decreased.In contrast,the expression of H19 was significantly down-regulated,while the expression of miR-29b-3p was up-regulated after GZFLW administration.

Figure 1.GZFLW downregulates H19 and upregulates miR-29b-3p.(Wu P,et al.,2023)