Witek Andrzej, Skałba Piotr, Paul Monika, Graniczka Michał, Mazurek Urszula, Chromy Grzegorz, Wilczok Tadeusz
Katedry i Kliniki Połoznictwa i Ginekologii SAM w Katowicach.
Ginekol Pol. 2003 Sep;74(9):897-902.
Estrogen target genes play essential role among pathogenetic and prognostic factors of endometrial adenocarcinoma; and among them estrogen receptor genes. Although prevailing estrogen receptor type is ER-alpha in endometrium, transduction of signal carried by estrogens can be also mediate by ER-beta, which biological meaning relies on modulation of ER-alpha transcriptional activity. There is hypothesized that ER-beta can keep under control mitogenic activity mediating by ER-alpha. The aim of the study was qualitative and quantitative analysis of wtER-beta and ER-beta/delta 5-6, ER-beta/delta 6 isoforms in endometrial adenocarcinoma. Material used in the study included specimens obtained from 27 female: group I--normal endometrium (n = 12), group II--endometrial adenocarcinoma (n = 15). RNA was extracted from the analyzed material using phenol-chloroform method by Total RNA Prep Plus. Qualitative analysis was performed basing on RT-PCR reaction and polyacrylamide gel electrophoreses. For all RNA extracts of the samples examined RT-PCR was performed with a sequence detector ABIPRISMTM7700--Perkin-Elmer Applied Biosystems (RT: 600 C-30 min; PCR: 950 C-5 min, 40 cycles: 950 C-30s, 600 C-1 min, and 720 C-10 min) in order to determine the number of RNA copies (which corresponds to the number of cDNA copies) for wtER-beta and ER-beta/delta 5-6 and ER-beta/delta 6. Specificity of QRT-PCR reaction was determined by delimitation of melting temperature of all examined amplimers (ABI PRISM7000, Qu-antiTect SYBR Green RT-PCR Kit) and by sequence analysis using ABI PRISM377 DNA Sequencer. Wild-type of estrogen receptor beta and ER-beta/delta 5-6, ER-beta/delta 6 isoforms--coming into alternative splicing of mRNA, presented both, in proliferative endometrium and endometrial adenocarcinoma. Copy number of wtER-beta mRNA was significantly (p < 0.05, Mann-Whitney U test) lower in comparison with proliferative endometrium and correlated with decrease of ER-beta/delta 6 mRNA copy number (r = 0.85; p < 0.05). Preliminary results confirming decrease of wtER-beta, ER-beta/delta 5/6 and ER-beta/delta 6 mRNA copy number in endometrial adenocarcinoma can show their relationship with high risk of carcinogenesis.
雌激素靶基因在子宫内膜腺癌的发病机制和预后因素中起着至关重要的作用；其中包括雌激素受体基因。尽管子宫内膜中占主导地位的雌激素受体类型是ER-α，但雌激素携带的信号转导也可由ER-β介导，其生物学意义依赖于对ER-α转录活性的调节。据推测，ER-β可以控制由ER-α介导的促有丝分裂活性。本研究的目的是对子宫内膜腺癌中的野生型ER-β、ER-β/δ5-6和ER-β/δ6亚型进行定性和定量分析。本研究使用的材料包括从27名女性身上获取的标本：第一组——正常子宫内膜（n = 12），第二组——子宫内膜腺癌（n = 15）。使用Total RNA Prep Plus通过苯酚-氯仿法从分析材料中提取RNA。基于RT-PCR反应和聚丙烯酰胺凝胶电泳进行定性分析。对于所检测样本的所有RNA提取物，使用序列检测仪ABIPRISMTM7700（珀金埃尔默应用生物系统公司）进行RT-PCR（逆转录：60℃-30分钟；聚合酶链反应：95℃-5分钟，40个循环：95℃-30秒，60℃-1分钟，72℃-10分钟），以确定野生型ER-β、ER-β/δ5-6和ER-β/δ6的RNA拷贝数（对应于cDNA拷贝数）。通过确定所有检测扩增子的熔解温度（ABI PRISM7000，QuantiTect SYBR Green RT-PCR试剂盒）以及使用ABI PRISM377 DNA测序仪进行序列分析来确定QRT-PCR反应的特异性。雌激素受体β的野生型以及ER-β/δ5-6和ER-β/δ6亚型——通过mRNA的可变剪接产生，在增殖期子宫内膜和子宫内膜腺癌中均有出现。与增殖期子宫内膜相比，野生型ER-β mRNA的拷贝数显著降低（p < 0.05，曼-惠特尼U检验），并且与ER-β/δ6 mRNA拷贝数的减少相关（r = 0.85；p < 0.05）。初步结果证实子宫内膜腺癌中野生型ER-β、ER-β/δ5/6和ER-β/δ6 mRNA拷贝数的减少可能表明它们与高致癌风险之间的关系。
