摘要
目的探究缺氧环境下影响子宫内膜腺上皮细胞增殖的潜在机制。方法分别在常氧和缺氧条件下(1%氧浓度)培养子宫内膜腺上皮细胞,提取RNA,高通量测序分析两种状态子宫内膜腺上皮细胞微小RNAs(microRNAs,miRNAs)的差异表达,并利用实时荧光定量PCR(qPCR)对差异表达的miRNA进行验证;通过Targetscan网站分析差异显著的miRNAs与增殖相关的靶基因,采用Western blot检测Wnt8b蛋白的表达。细胞计数(CCK8)检测正常和缺氧条件下培养子宫内膜腺上皮细胞之间的细胞增殖的情况。结果miRNA高通量测序结果显示缺氧条件下miR-7851-3p、miR-125a-3p和miR-141-5p等16个miRNAs在子宫内膜腺上皮细胞中表达升高,qPCR检测结果显示缺氧环境下子宫内膜腺上皮细胞的miR-7851-3p的表达显著升高(P<0.001)。靶基因预测结果显示Wnt8b是miR-7851-3p潜在靶基因,而在缺氧环境下子宫内膜腺上皮细胞的Wnt8b蛋白表达降低。CCK8检测结果显示缺氧条件下子宫内膜腺上皮细胞增殖能力降低,尤其在培养36 h时差异显著(P<0.001)。结论子宫内膜腺上皮细胞在缺氧环境下miR-7851-3p表达上调,可能通过抑制Wnt8b蛋白的表达而抑制子宫内膜腺上皮细胞的增殖。
Objective:To investigate the potential mechanism of endometrial glandular epithelial cell proliferation under hypoxic environment.Methods:Endometrial glandular epithelial cells were cultured under normoxic or hypoxic condition(1%oxygen concentration),and the total RNAs were extracted.High throughput sequencing was used to analyze the differential expression of microRNAs(miRNAs)in endometrial glandular epithelial cells under two conditions,and real-time fluorescent quantitative PCR(qPCR)was used to verify the differential expression of miRNAs.The target genes related to proliferation were analyzed by Targetscan website,and the expression of Wnt8b protein was detected by Western blot.Cell count(CCK8)was used to detect the cell proliferation of endometrial glandular epithelial cells under normoxic or hypoxia condition.Results:MicroRNAs high throughput sequencing results showed that the expressions of 16 miRNAs,such as miR-7851-3p,miR-125a-3p and miR-141-5p and miR-141-5p,etc.were increased in endometrial glandular epithelial cell under hypoxic environment.qPCR results showed that the expressions of miR-7851-3p was significantly increased in endometrial glandular epithelial cells under hypoxic environment(P<0.001).The results of target gene prediction showed that Wnt8b was a potential target gene of mir-7851-3p,and the expression of Wnt8b protein in endometrial glandular epithelial cells decreased under hypoxic environment.The results of CCK8 showed that the proliferation of endometrial glandular epithelial cells decreased under hypoxic environment,especially at 36 hours(P<0.001).Conclusions:The expression of miR-7851-3p in endometrial glandular epithelial cells was up-regulated under hypoxic environment,which may inhibit the proliferation of endometrial glandular epithelial cells by suppressing the expression of Wnt8b protein.
作者
王含必
窦帅杰
刘思邈
张婉玉
刘美芝
邓成艳
WANG Han-bi;DOU Shuai-jie;LIU Si-miao;ZHANG Wan-yu;LIU Mei-zhi;DENG Cheng-yan(Centre of Gynecological Endocrinology&Assisted Reproduction,National Clinical Research Center for Obstetric&Gynecologic Diseases,State Key Laboratory of Complex Severe&Rare Diseases,Peking Union Medical College Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100730;Beijing Zhicheng Biomedical Technology Co.,LTD,Beijing 100176)
出处
《生殖医学杂志》
CAS
2022年第9期1253-1258,共6页
Journal of Reproductive Medicine
基金
中华医学会临床医学科研专项资金“干细胞基础研究”项目(19020020781)
中国医学科学院中央级公益性科研院所基本科研业务费专项资金(2021-PT320-001)。
关键词
子宫内膜腺上皮细胞
微小核糖核酸
信号通路
细胞增殖
Endometrial glandular epithelial cells
MicroRNAs
Signaling pathway
Cell proliferation
