MiR-296-3p调节多囊卵巢综合征大鼠颗粒细胞凋亡的作用机制

doi:10.13523/j.cb.2303008

中国生物工程杂志

2023, Vol. 43

Issue (4): 20-29 DOI: 10.13523/j.cb.2303008

研究报告

MiR-296-3p调节多囊卵巢综合征大鼠颗粒细胞凋亡的作用机制

白雪¹,于新月^1,^*(

),郑春杨²,侯聪³,李敏⁴

1.北部战区总医院沈阳 110052
2.沈阳菁华医院有限公司沈阳 110001
3.辽宁汇智同达生物科学有限责任公司沈阳 110034
4.北京中源合聚生物科技有限公司北京 100016

Mechanism of MiR-296-3p Regulating Granulosa Cell Apoptosis in Rats with Polycystic Ovary Syndrome

BAI Xue¹,YU Xin-yue^1,^*(

),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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摘要：

目的:探讨miR-296-3p对多囊卵巢综合征(polycystic ovary syndrome,PCOS)大鼠颗粒细胞凋亡的影响。方法:全自动化学发光免疫分析法检测PCOS大鼠血清雌二醇(estradiol,E2)、孕酮(progesterone,P)、睾酮(testosterone,T)水平;利用苏木素-伊红染色(hematoxylin-eosin staining,HE)检测卵巢病理学改变;TUNEL法检测卵巢细胞凋亡;qPCR检测卵巢miR-296-3p表达水平;将各质粒分别转染卵巢颗粒细胞(ovarian granulosa cells,GCs)后分为miR-NC、miR-296-3p mimic、miR-296-3p inhibitor、miR-296-3p mimic+Vector、miR-296-3p mimic+PRKCA和miR-296-3p mimic+PRKCA+NF-κB组,以未处理的GCs为对照组;Western blot法检测p38、p-p38及NF-κB的表达水平,利用StarBase在线预测和双荧光素酶报告实验验证miR-296-3p与PRKCA的靶向关系,流式细胞术检测细胞凋亡率。结果:与对照组相比,PCOS大鼠卵巢中E2、P、T水平升高,细胞凋亡比例增加,miR-296-3p表达升高;miR-296-3p与PRKCA具有靶向关系,在3'UTR区存在结合位点;与miR-NC组相比,miR-296-3p mimic组PRKCA mRNA表达水平显著下调,p-p38/p38和NF-κB表达上调,miR-296-3p inhibitor组PRKCA mRNA表达水平显著上调,p-p38/p38和NF-κB表达下调;与miR-NC组相比,miR-296-3p mimic组和miR-296-3p mimic+Vector组细胞凋亡比例显著上调;与miR-296-3p mimic组和miR-296-3p mimic+Vector组相比,miR-296-3p mimic+PRKCA组卵巢颗粒细胞凋亡比例显著下调;与miR-296-3p mimic+PRKCA组相比,miR-296-3p mimic+PRKCA+NF-κB组卵巢颗粒细胞凋亡比例显著上调。结论:miR-296-3p通过靶向PRKCA激活p38 MAPK/NF-κB信号通路介导多囊卵巢综合征大鼠颗粒细胞凋亡。

关键词： 多囊卵巢综合征; miR-296-3p; 蛋白激酶Cα; 卵巢颗粒细胞; 细胞凋亡

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 words: Polycystic ovarian syndrome MiR-296-3p Protein kinase C alpha(PRKCA) Granular cells of ovary Apoptosis

收稿日期: 2023-03-02 出版日期: 2023-05-04

ZTFLH:

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通讯作者: *电子信箱:bxzhk5588@163.com

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引用本文:

白雪, 于新月, 郑春杨, 侯聪, 李敏. MiR-296-3p调节多囊卵巢综合征大鼠颗粒细胞凋亡的作用机制[J]. 中国生物工程杂志, 2023, 43(4): 20-29.

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.

链接本文:

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

图1 两组大鼠血清生殖激素水平比较

图2 各组大鼠卵巢组织HE染色切片

图3 各组大鼠卵巢组织TUNEL染色图

图4 各组大鼠卵巢中miR-296-3p的表达

图5 双荧光素酶报告实验检测miR-296-3p与PRKCA的靶向关系

图6 卵巢颗粒细胞中PRKCA的表达

图7 卵巢颗粒细胞中p-p38/p38和NF-κB的表达 Expression of p-p38/p38和NF-κB in cumulus granulosa cells in each group

图8 各组卵巢颗粒细胞凋亡百分比

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