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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 and clone the gene fragments specific to the pathogenesis of endometrial carcinoma and to explore the molecular mechanism of endometrial carcinoma. Methods Human endometrial stromal epithelial cells and endometrial carcinoma cells were obtained by laser capture microdissection (LCM). MicroRNAs were extracted and purified by enrichment. Fluorescent differential display (FDD- PCR) were used to screen differentially expressed genes specific for the pathogenesis of endometrial cancer. Reverse Northern blotting (using amplified RNA) was used to validate the differential fragments. The positive fragments were analyzed by BLAST using GenBank. Results A total of 38 differential bands were obtained, of which 3 were highly expressed in normal endometrium and 35 were highly expressed in endometrial cancer. The 10 differential bands recovered and sequenced were cloned and sequenced. Reverse Northern blotting was used to identify 6 positive differential gene fragments. The BLAST alignment showed that the homology of L1.1 to CDK7 was 99%. The homology of L1.9 to human protein phosphatase 1 regulatory subunit 12A (PPP1R12A) was 99 %; L1.21, L1.22 has 100% homology with the suppressor of E1A activating gene 1 (CREG); L1.25, L1.26 is homologous to the zinc transporter 10 (SLC39A10) in the zinc transporter family Sex is above 98%. Conclusion Through the combination of LCM and FDD-PCR technology, we obtained the gene fragments that are specific to the pathogenesis of endometrial cancer. For the first time from the gene level found in CDK7, PPP1R12A, CREG, SLC39A10 and the incidence of endometrial cancer correlation, complement the understanding of the molecular mechanism of the pathogenesis of endometrial cancer. Which CDK7, CREG, SLC39A10 can be used as a new candidate endometrial cancer oncogene, PPP1R12A can be used as a new endometrial cancer candidate tumor suppressor gene for further research.