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中国生物工程杂志

China Biotechnology
China Biotechnology  2023, Vol. 43 Issue (4): 20-29    DOI: 10.13523/j.cb.2303008
    
Mechanism of MiR-296-3p Regulating Granulosa Cell Apoptosis in Rats with Polycystic Ovary Syndrome
BAI Xue1,YU Xin-yue1,*(),ZHEN Chun-yang2,HOU Cong3,LI Min4
1. General Hospital of Northern Theater Command, Shenyang 110052, China
2. Shenyang Jinghua Hospital Co., Ltd., Shenyang 110001, China
3. Liaoning Huizhi Tongda Biological Science Co., Ltd., Shenyang 110034, China
4. Beijing Zhongyuan Co., Ltd., Beijing 100016, China
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Abstract  

Objective: To investigate the effect of miR-296-3p on granulosa cell apoptosis in rats with polycystic ovary syndrome (PCOS). Methods: The levels of estradiol (E2), progesterone (P) and testosterone (T) in serum of PCOS rats were detected by automatic chemiluminescence immunoassay; HE staining was used to detect the pathological changes of the ovary; TUNEL was used to detect apoptosis in the ovary; The expression level of miR-296-3p and PRKCA in the ovary was detected by qPCR; Ovarian granulosa cells (GCs) were divided into miR-NC, miR-296-3p mimic, miR-296-3p inhibitor, miR-296-3p mimic+Vector, miR-296-3p mimic+PRKCA and miR-296-3p mimic+PRKCA+NF-κB groups after transfection of various plasmids, and untreated GCs were used as control group. The expression level of p38, p-p38 and NF-κB was detected by Western blot. StarBase website and double luciferase report test predicted and verified the targeting relationship between miR-296-3p and PRKCA, respectively. Flow cytometry was used to detect cell apoptosis rate. Results: Compared with the control group, the levels of E2, P and T in the ovaries of PCOS rats increased; The proportion of apoptosis increased; The expression of miR-296-3p increased. miR-296-3p has a targeting relationship with PRKCA, and there are binding sites in the 3'UTR region. Compared with miR-NC group, the expression level of PRKCA mRNA in miR-296-3p mimic group was significantly decreased, and p-p38/p38 and NF-κB expression was up-regulated. Compared with miR-NC group, the expression level of PRKCA mRNA in miR-296-3p inhibitor group was significantly increased, and p-p38/p38 and NF-κB expression was down-regulated. Compared with the Mir-NC group, the percentage of apoptosis of miR-296-3p mimic and miR-296-3p mimic+Vector groups was significantly up-regulated. Compared with miR-296-3p mimic and miR-29-3p mimic+Vector, the percentage of apoptosis of ovarian granulocyte cells in miR-296-3p mimic+PRKCA group was significantly down-regulated. Compared with miR-296-3p mimic+PRKCA+NF-κB group, the percentage of apoptosis of ovarian granulosa cells in miR-296-3p mimic+PRKCA+NF-κB group was significantly up-regulated. Conclusion: MiR-296-3p activates p38 MAPK/NF-κB signal pathway by targeting PRKCA and mediating granulosa cell apoptosis in rats with polycystic ovary syndrome.



Key wordsPolycystic ovarian syndrome      MiR-296-3p      Protein kinase C alpha(PRKCA)      Granular cells of ovary      Apoptosis     
Received: 02 March 2023      Published: 04 May 2023
ZTFLH:  R285  
Cite this article:

BAI Xue, YU Xin-yue, ZHEN Chun-yang, HOU Cong, LI Min. Mechanism of MiR-296-3p Regulating Granulosa Cell Apoptosis in Rats with Polycystic Ovary Syndrome. China Biotechnology, 2023, 43(4): 20-29.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2303008     OR     https://manu60.magtech.com.cn/biotech/Y2023/V43/I4/20

Fig.1 Comparison of serum reproductive hormone levels between two groups of rats (a) Estradiol content in the serum (b) Progesterone content in the serum (c) Testosterone content in the serum. ** P<0.05
Fig.2 Representative images of HE staining of ovarian sections from a single rat in each group
Fig.3 Representative images of TUNEL staining of ovarian sections from a single rat in each group (a) Representative micrographs of TUNEL staining in each group (b) Quantification analysis of TUNEL staining in each group ** P<0.05
Fig.4 Expression of miR-296-3p in ovarian of rats in each group ** P<0.05
Fig.5 Targeting relationship between miR-296-3p and PRKCA detected by double luciferase report test * P < 0.05
Fig.6 Expression of PRKCA in cumulus granulosa cells in each group ** Compare with miR-NC group, P < 0.05
Fig.7 (a) Representative western blot bands (b) Quantification of relative protein expression ** Compare with miR-NC group, P < 0.05
Fig.8 Percentage of apoptosis in ovarian granulosa cells in each group (a) Apoptosis was measured by flow cytometry (b) Apoptosis rate statistics. ** Compare with miR-NC group, P < 0.05; ## Compare with miR-296-3p mimic+Vector group, P < 0.05; && Compare with miR-296-3p mimic+PRKCA group, P < 0.05
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