pLVX-EGFP-C1

Case Study 1

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)

Case Study 2

GZFLW regulates H19/miR-29-3p to inhibit autophagy.

Guizhi Fuling Wan inhibits autophagy of granulosa cells in polycystic ovary syndrome mice via H19/miR-29b-3p

Paper 2023

Gynecological Endocrinology | DOI

Background
This study explored whether Guizhi Fuling Wan (GZFLW) inhibits granulosa cell autophagy in polycystic ovary syndrome (PCOS) via the H19/miR-29b-3p pathway. PCOS model and control granulosa cells were treated with GZFLW-containing serum. qRT-PCR, luciferase assays, western blot, MDC staining, and mRFP-eGFP-LC3 imaging assessed H19, miR-29b-3p, PTEN, MMP-2, Bax, and autophagy. GZFLW downregulated H19, upregulated miR-29b-3p, reduced PTEN/MMP-2/Bax, and decreased autophagosomes. Inhibiting miR-29b-3p or overexpressing H19 reversed these effects, confirming that GZFLW suppresses PCOS granulosa cell autophagy through H19/miR-29b-3p.
Key Findings
GZFLW treatment significantly downregulated H19 and upregulated miR-29b-3p in PCOS granulosa cells (p<.01). Luciferase assays confirmed H19 as a direct target of miR-29b-3p and verified binding sites for miR-29b-3p in PTEN, MMP-2, and Bax. GZFLW reduced PTEN, MMP-2, and Bax protein levels in PCOS cells (p<.01). MDC staining and mRFP-eGFP-LC3 imaging showed that PCOS granulosa cells had increased autophagic vesicles, autophagosomes, and autophagic polymers, while GZFLW treatment markedly decreased these autophagy markers (p<.01). Inhibition of miR-29b-3p or overexpression of H19 reversed GZFLW's suppression of autophagy and attenuated its effects on PTEN, MMP-2, and Bax (p<.05 or p<.01). Thus, GZFLW inhibits excessive granulosa cell autophagy in PCOS via the H19/miR-29b-3p pathway, providing a potential therapeutic mechanism for PCOS.
Contribution
Creative Biogene supplied the pLVX-EGFP-C1 plasmid used in this study. This lentiviral vector likely facilitated H19 overexpression experiments, which were critical for showing that H19 reverses GZFLW-induced autophagy inhibition. Therefore, Creative Biogene provided an essential molecular tool for interrogating the H19/miR-29b-3p pathway, though the company did not participate in study design, data collection, or analysis.