摘要目的 探讨与雌激素受体α(Erα)和孕激素受体(PR)表达相关的微小RNA(microRNA,miRNA)在Ⅰ型和Ⅱ型内膜癌中的差异表达.方法 通过对两种细胞系Ishikawa和KLE细胞的裸鼠移植瘤组织进行病理形态学观察、免疫组化法检测其Erα、PR和p53蛋白的表达以及活细胞计数(CCK-8)法检测雌、孕激素作用后Ishikawa和KLE细胞的生长情况,对Ishikawa和KLE细胞进行组织学分型及鉴定;对无雌、孕激素环境下培养的lshikawa和KLE细胞,用miRNA微阵列芯片筛选Ishikawa和KLE中差异表达的miRNA;用miRANDA和TargetScan软件结合芯片筛选结果,预测Ishikawa和KLE细胞中可能以ESRI(翻译产物为Erα蛋白)和PGR(翻译产物为PR蛋白)为靶基因的miRNA;用实时荧光定量PCR技术验证在体内和体外培养的lshikawa和KLE细胞中以及10例内膜癌患者癌组织标本中其表达的差异性.结果 经组织学分型及鉴定显示,lshikawa细胞来源于Ⅰ型内膜癌,KLE细胞来源于Ⅱ型内膜癌;miRNA微阵列芯片筛选出Ishikawa和KLE细胞中差异表达的miRNA共126个;可能以ESRI为靶基因的miRNA为has-miR-99a与has-miR-100,可能以PGR为靶基因的miRNA为has-miR-378与has-miR-768-3p;实时荧光定量PCR技术验证显示,has-miR-100、99a、378、768-3p在体外和体内培养的Ishikawa和KLE细胞中的差异表达与miRNA微阵列芯片筛选的结果是一致的;Ⅰ型内膜癌组织中has-miR-100的表达明显低于Ⅱ型内膜癌组织(P＜0.01).结论 has-miR-100在Ⅱ型内膜癌组织中的表达明显高于Ⅰ型内膜癌,其靶基因可能为ESR1.

abstractsObjective To identify differentially expression of microRNAs associated with expression of estrogen receptor α(ERα)and progesterone receptor(PR)between type Ⅰ and type Ⅱ endometrial adenocarcinoma.Methods Two kinds of endometrial adenocarcinoma cell lines,Ishikawa and KLE,was transplanted into node mice and biopsied to identify the expression of ERα,PR and p53,and test their response to estrogen and progesterone.Cultured the two cell lines under the estrogen-free and progesteronefree circumstance,total RNA was isolated to identify the differentially expressed microRNAs by microarray for prediction the microRNAs which target ESR1 and PGR by software miRANDA and TargetScan,and then was validated by real-time PCR in two cell lines cultured both in vivo and in vitro and ten specimens from patients.Results Ishikawa cell line was confirmed from type Ⅰ endometrial adenocarcinoma.KLE cell line was confirmed from typeⅡendometrial adenocarcinoma.One hundred and twenty-six differentially expressed microRNAs between the two cell lines were identified by mieroRNA microarray,among of which may target ESR1 inchded hsa-miR-100,99a,and may tgrget PGR included hsa-miR-378,768-3p-The differential expression of hsa-miR-100,99a,378,768-3p identified by microarray between Ishikawa and KLE in vivo and in vitro was equal to that by real-time PCR,while Hsa-miR-100 was significantly down expressed in type Ⅰ group specimens compared to type Ⅱ group(P＜0.01).Conclusion Hsa-miR-100 is significantly down-expressed in type Ⅰ endometrial adenocarcinoma compared to type Ⅱ,which may be a great potential to target ESR1.