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孕激素诱导的microRNA-1a抑制子宫内膜上皮细胞增殖

潘俊丽, 袁东智, 聂莉, 张金虎, 岳利民^*

四川大学华西基础医学与法医学院生理学教研室，成都 610041

摘要

本文旨在研究孕激素诱导microRNA-1a (miR-1a)在子宫内膜上皮细胞(endometrial epithelial cells, EECs)的表达及其对EECs增殖的作用。用雌激素(E2组)或雌、孕激素联合(E2P4组)处理去卵巢小鼠，qPCR检测miR-1a的成熟体miR-1a-3p的表达；将miR-1a-3p拟似剂或抑制剂注入各组小鼠的一侧子宫角，另一侧注入拟似剂或抑制剂对照，流式细胞术检测miR-1a-3p对EECs细胞周期的影响；免疫组化检测miR-1a-3p对细胞周期蛋白cyclin D2、cyclin E1和cyclin E2表达的影响。在体外实验，将分离培养的原代小鼠EECs分为两组，分别给予雌激素处理(E2组)和雌、孕激素联合处理(E2P4组)，qPCR检测miR-1a-3p的表达；再用miR-1a-3p拟似剂作用于雌激素预处理细胞，对照组给予非特异性拟似剂，EdU掺入实验检测细胞增殖活性。体内qPCR结果显示，E2P4组小鼠EECs miR-1a-3p的表达量相比E2组明显增加(P < 0.05)。流式细胞术结果显示miR-1a-3p拟似剂能够将E2处理细胞的细胞周期进程阻滞在G1/S期(P < 0.05)；miR-1a-3p抑制剂能够部分逆转P4对细胞周期进程的G1/S期阻滞(P < 0.05)。小鼠子宫组织免疫组化结果显示miR-1a-3p拟似剂能较明显地抑制cyclin E1和cyclin E2蛋白的表达(P < 0.05)，而对cyclin D2蛋白表达无影响(P > 0.05)。体外qPCR结果显示E2处理组和E2P4联合处理组细胞中都未检测到miR-1a-3p表达，体外EdU掺入实验表明外源性miR-1a-3p拟似剂能够一定程度抑制雌激素促细胞增殖作用(P < 0.05)。以上结果表明，孕激素可通过诱导miR-1a-3p表达引起细胞周期G1/S期阻滞，从而抑制EECs细胞增殖，这种抑制增殖作用与其抑制cyclin E1和cyclin E2蛋白表达有关。

关键词： 孕激素; miR-1a; 子宫内膜上皮细胞; 细胞增殖

分类号：Q4

[Progesterone-induced microRNA-1a inhibits the proliferation of endometrial epithelial cells.] [Article in Chinese]

PAN Jun-Li, YUAN Dong-Zhi, NIE Li, ZHANG Jin-Hu, YUE Li-Min^*

Department of Physiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China

Abstract

The aim of the present study was to investigate the effects of progesterone (P4)-induced microRNA-1a (miR-1a) on the proliferation of endometrial epithelial cells (EECs) and the underlying mechanism. In vivo, following subcutaneous injection of estradiol (E2) alone (E2 group) or combined injections of E2 and P4 (E2P4 group) in ovariectomized mice, quantitative real-time PCR (qPCR) was used to check the expression of miR-1a-3p in the directly isolated mouse EECs. The agomir or antagomir specific for miR-1a-3p was injected into one side of the uterine horns of ovariectomized mice pretreated with E2 alone or in combination with P4, and the non-specific control agomir or antagomir was injected into their contralateral horns. Flow cytometry was used to analyze the cell cycle of EECs. Immunohistochemistry (IHC) was used to examine the location and expression of cyclin D2, cyclin E1, and cyclin E2 in the uterine tissue sections. In vitro, primary cultured mouse EECs were pretreated with E2 alone (E2 group) or in combination with P4 (E2P4 group). qPCR was used to detect the expression of miR-1a-3p. Exogenous mimic of miR-1a-3p was transfected into E2-pretreated EECs, and EdU incorporation analysis was used to test the proliferation activity of the EECs. The result of in vivo experiment showed that the expression of miR-1a-3p in E2P4 group was significantly higher than that in E2 group (P < 0.05). The miR-1a-3p agomir arrested cell cycle at G1 to S transition in the mice injected subcutaneously with E2 alone (P < 0.05). Conversely, silencing of miR-1a-3p with transfection of miR-1a-3p antagomir promoted the entry of cells into S phase in the mice injected subcutaneously with both E2 and P4 (P < 0.05). The expressions of cyclin E1 and cyclin E2, except for cyclin D2, in uterine sections were also dramatically reduced by miR-1a-3p overexpression in the uterine epithelium (P < 0.05). In vitro, miR-1a-3p was not expressed in the cells of both E2 and E2P4 groups. The mimic of miR-1a-3p decreased EECs proliferation activity (P < 0.05). These results indicate that P4-induced miR-1a can inhibit the expression of cyclin E1 and cyclin E2, consequently suppressing the proliferation of mouse EECs by arresting cells at G1/S phase.

Key words: progesterone; microRNA-1a; endometrial epithelial cells; cell proliferation

收稿日期：2016-04-06　　录用日期：2016-07-01

通讯作者：岳利民　　E-mail: yuelimin@scu.edu.cn

引用本文：

潘俊丽, 袁东智, 聂莉, 张金虎, 岳利民. 孕激素诱导的microRNA-1a抑制子宫内膜上皮细胞增殖[J]. 生理学报 2016; 68 (6): 716-724.

PAN Jun-Li, YUAN Dong-Zhi, NIE Li, ZHANG Jin-Hu, YUE Li-Min. [Progesterone-induced microRNA-1a inhibits the proliferation of endometrial epithelial cells.] [Article in Chinese]. Acta Physiol Sin 2016; 68 (6): 716-724 (in Chinese with English abstract).