人子宫内膜癌相关基因的研究

Screening,cloning and identification of the human endometrial carcinoma-related genes

目的筛选、克隆与鉴定人子宫内膜癌发病特异相关的基因片段,探索子宫内膜癌发生的分子机制。方法运用激光捕获显微切割技术(LCM)获取无间质混杂的人正常子宫内膜腺管上皮细胞与子宫内膜癌细胞,提取微量RNA并进行纯化及浓缩,采用荧光差异显示技术(FDD-PCR)筛选与子宫内膜癌发病特异相关的差异基因片段,反向Northern点杂交(使用扩增的RNA)验证差异片段,对阳性片段利用GenBank进行BLAST比对分析。结果共获得38条差异条带,其中3条在正常子宫内膜中高表达,35条在子宫内膜癌中高表达。对10条再回收差异条带进行克隆并测序,经反向Northern点杂交鉴定,获得6条阳性差异基因片段。经BLAST比对分析显示,L1.1与细胞周期蛋白依赖性激酶7(CDK7)同源性为99%;L1.9与人类蛋白磷酸酶1调节抑制亚单位12A (PPP1R12A)同源性为99%;L1.21、L1.22与E1A激活基因1的抑制子(CREG)同源性为100%;L1.25、L1.26与锌转运家族中的10号锌转运体(SLC39A10)同源性为98%以上。结论通过LCM技术与FDD-PCR技术的结合,获得了与子宫内膜癌发病特异相关的基因片段。首次从基因水平发现了CDK7、PPP1R12A、CREG、SLC39A10与子宫内膜癌发病的相关性,补充了对子宫内膜癌发病的分子机制的认识。其中CDK7、CREG、SLC39A10可以作为新的子宫内膜癌候选癌基因,PPP1R12A可以作为新的子宫内膜癌候选抑癌基因来进行更深层次的研究。

Objective To screen, clone and identify the eDNA fragments of human endometrial carcinoma-related genes,and explore the molecular mechanism of endometrial carcinogenesis. Methods Pure endometrial glandular epithelial cells and endometrial carcinoma cells were obtained by laser capture microdissection （LCM）. RNA from these cells was isolated, and differentially expressed gene fragments that were specialy relevant to endometrial carcingenesis were identified by using fluorescence differential display reverse transcription polymerase chain reaction （FDD-PCR）. The selected fragments were cloned, sequenced and verified by reverse Northern blot analysis, and positive fragments were BLAST analysed and compared with those in Genbank. Results 38 differential fragments were isolated, 3 of which were expressed more abundantly in normal endometrium and 35 were highly expressed in endometrial carcinoma. 10 fragments were recoverd, cloned and sequenced, confirmed by reverse Northern blot analysis, among which 6 fragments were positive. BLAST analysis showed that T1.1 was homologous to cyclin-dependent protein kinase 7 （ CDK7, 99% ）; L1.9 was homologous to protein phosphatase 1 regulatory （inhibitor） subunit 12A （PPP1R12A, 99% ） ; L1.21 and L1.22 were homologous to cellular repressor of E1A-stimulated genes 1 （ CREG, 100% ） ; L1. 25 and L1. 26 were homologous to solute carrier family 39 （zinc transporter ） member 10 （SLC39A10, 〉 98% ）. Conclusion Gene fragments related to endometrial carcinoma have been obtained by applying LCM and FDD-PCR. To our knowledge it is the first time that the correlation between CDK7, PPP1R12A, CREG, SLC39A10 and endometrial carcinoma is discovered at mRNA level, and their role in molecular mechanism of cancinogenesis is discussed. CDK7, CREG, SLC39A10 as new candidate oncogene and PPP1R12A as new candidate anti-oncogene are worthy of being further investigated in the future.