摘要
目的探究缺氧状态下的子宫内膜腺上皮细胞(EEC)增殖和迁移的可能机制。方法分别提取缺氧(1%氧浓度,缺氧组)和常氧(21%氧浓度,常氧组)条件下EEC的RNA进行高通量测序(RNA-sequence),分析两组细胞中微小RNA(microRNA,miRNA)的差异表达。利用实时荧光定量PCR(RT-qPCR)的方法对差异表达的miRNA即has-miR-215-5p进行鉴定,通过miRNA靶基因数据库网站(Target Scan)分析has-miR-215-5p的靶基因。Western Blot检测两组细胞中靶基因膀胱癌相关蛋白(BLCAP)的蛋白表达水平,细胞计数实验检测两组EEC的增殖情况,划痕实验检测两组EEC的迁移能力。结果测序结果显示,缺氧组EEC中has-miR-215-5p表达显著上升(P<0.001),RT-qPCR验证结果与测序结果一致。miRNA Target Scan数据库检索结果显示,BLCAP是has-miR-215-5p的下游靶基因,双荧光素酶报告基因实验结果显示has-miR-215-5p与BLCAP-3’-UTR结合,结合位点突变后结合活性消失。Western Blot结果显示,与常氧组相比,缺氧组EEC中的BLCAP蛋白表达显著下降(P<0.001)。CCK-8及划痕实验分别提示与常氧组相比,缺氧组的EEC增殖能力显著降低、迁移能力显著增强(P<0.001)。结论缺氧条件下EEC中has-miR-215-5p可能通过与BLCAP-3’-UTR结合,抑制BLCAP的表达活性,导致EEC的增殖降低与迁移增强。
Objective:To explore the potential mechanism of endometrial glandular epithelial cell(EEC)proliferation and migration under hypoxia condition.Methods:RNA from EEC under hypoxia(1%oxygen concentration)and normoxia(21%oxygen concentration)conditions were extracted for RNA-sequence to analyze the differences in intracellular microRNA(miRNA)gene expression between the two status.Reverse transcription quantitative polymerase chain reaction(RT-qPCR)was used to identify differentially expressed miRNA(has-miR-215-5p).The target genes of has-miR-215-5p were analyzed through miRNA target gene database website(Target Scan).Western blot was used to detect the expression of bladder cancer-associated protein(BLCAP).Cell counting kit-8(CCK-8)was used to observe the proliferation of EEC and the scratch test was used to detect the migration ability of EEC under hypoxia and normoxia conditions.Results:The miRNA sequencing results showed that the expression of has-miR-215-5p in EEC was significantly increased under hypoxia conditions(P<0.001),and the RT-qPCR result was the same as the sequencing results.The miRNA Target Scan database was searched to analyze and predict that BLCAP was the downstream target gene of has-miR-215-5p.The results of dual-luciferase reporter assay showed that has-miR-215-5p binded to BLCAP-3′-UTR,and the binding activity disappeared after the mutation of the binding site.Western blot analysis confirmed that the expression of BLCAP protein in EEC was significantly decreased under hypoxia conditions(P<0.001).CCK-8 and scratch experiments showed that the proliferation ability of EEC was significantly reduced and the migration ability was significantly enhanced under hypoxia conditions(P<0.001).Conclusions:Under hypoxia conditions,has-miR-215-5p in EEC may inhibit the expression activity of BLCAP by binding to BLCAP-3′-UTR,resulting in decreased proliferation and enhanced migration of EEC.
作者
张婉玉
王含必
邓成艳
ZHANG Wan-yu;WANG Han-bi;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&Peking Union Medical College,Beijing 100730)
出处
《生殖医学杂志》
CAS
2024年第4期494-500,共7页
Journal of Reproductive Medicine
基金
北京协和医院中央高水平医院临床科研专项项目(2022-PUMCH-B-080,2022-PUMCH-C-64)。
