Mechanism study of miR-29a-3p targeting SGMS2 to regulate chronic inflammation in PCOS ovarian granulosa cells
10.3760/cma.j.cn101441-20220729-00317
- VernacularTitle:miR-29a-3p靶向 SGMS2调控PCOS卵巢颗粒细胞慢性炎症的机制研究
- Author:
Fang XU
1
;
Xiaoke LI
;
Zimeng PAN
;
Jinling LIANG
;
Ming MA
;
Hongying KUANG
;
Miao SUN
Author Information
1. 黑龙江中医药大学附属第一医院妇科，哈尔滨 150040
- Publication Type:Journal Article
- Keywords:
Polycystic ovary syndrome;
miR-29a-3p;
Sphingomyelin synthase 2;
Ovarian granulosa cells;
Inflammation
- From:
Chinese Journal of Reproduction and Contraception
2023;43(10):1032-1040
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the regulatory mechanism of miR-29a-3p/ SGMS2 axis on the inflammatory response of ovarian granulosa cells (KGN). Methods:By constructing and validating overexpression and knockdown of miR-29a-3p and SGMS2 transfected KGN cell models, dual luciferase reporter gene assay was used to detect the binding of miR-29a-3p to SGMS2. MTT assay was used for detecting the cell proliferation. Fluorescence intensity of proliferating cell nuclear antigen (PCNA) and Ki67 protein were detected by immunofluorescence. Apoptosis was detected by flow cytometry. The expression levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β and sphingomyelin in cell supernatant were detected by enzyme-linked immunosorbent assay. The protein expression levels of Caspase-3, cleaved-Caspase-3, SGMS2 and p-p65 in KGN were detected by Western blotting. Results:miR-29a-3p had site-specific binding to SGMS2 and can negatively regulated SGMS2 expression level. Overexpression of SGMS2 in KGN cells was able to increase their levels of absorbance ( P=0.007) and enhanced the immunofluorescence intensity of the protein of PCNA and Ki67 (all P<0.001). Overexpression of SGMS2 could reduce the apoptosis rate of KGN cells ( P=0.001). Overexpression of SGMS2 increased the expression levels of TNF-α, IL-6, IL-1β and sphingomyelin (all P<0.001). The expressions of cleaved-Caspase-3, p-p65 and SGMS2 proteins were elevated in KGN cells when SGMS2 was overexpressed ( P=0.001, P<0.001, P<0.001), while knock down SGMS2, the above results had a trend of change opposite to overexpression of SGMS2. Conclusion:There is a targeted negative regulatory relationship between miR-29a-3p and SGMS2, which can promote the inflammatory response of KGN and may provide a target for the treatment of polycystic ovary syndrome.
