Pinto Rita, Barros Rita, Pereira-Castro Isabel, Mesquita Patricia, da Costa Luis T, Bennett Eric P, Almeida Raquel, David Leonor
1] Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal [2] Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal [3] Faculty of Medicine, University of Porto, Porto, Portugal.
1] Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal [2] Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal.
Lab Invest. 2015 Jul;95(7):718-27. doi: 10.1038/labinvest.2015.52. Epub 2015 Apr 13.
De novo expression of Sialyl-Tn (STn) antigen is one of the most common features of intestinal metaplasia (IM) and gastric carcinomas, and its biosynthesis has been mostly attributed to ST6GalNAc-I activity. However, the regulation of this glycosyltransferase expression is not elucidated. In IM lesions and in the intestine, CDX2 homeobox transcription factor is co-expressed with STn and ST6GalNAc-I. We therefore hypothesized that CDX2 might induce STn expression by positive regulation of ST6GalNAc-I. We showed that ST6GalNAc-I transcript levels and CDX2 have a coordinated expression upon Caco-2 in vitro differentiation, and overexpression of CDX2 in MKN45 gastric cells increases ST6GalNAc-I transcript levels. Nine putative CDX-binding sites in the ST6GalNAc-I-regulatory sequence were identified and analyzed by chromatin immunoprecipitation in Caco-2 cells and in IM. The results showed that CDX2 protein is recruited to all regions, being the most proximal sites preferentially occupied in vivo. Luciferase assays demonstrated that CDX2 is able to transactivate ST6GalNac-I-regulatory region. The induction was stronger for the regions mapped in the neighbourhood of ATG start codon and site-directed mutagenesis of these sites confirmed their importance. In conclusion, we show that CDX2 transcriptionally regulates ST6GalNAc-I gene expression, specifically in the preneoplastic IM lesion.
唾液酸-Tn（STn）抗原的从头表达是肠化生（IM）和胃癌最常见的特征之一，其生物合成主要归因于ST6GalNAc-I的活性。然而，这种糖基转移酶表达的调控机制尚未阐明。在IM病变和肠道中，CDX2同源框转录因子与STn和ST6GalNAc-I共同表达。因此，我们推测CDX2可能通过正向调控ST6GalNAc-I来诱导STn表达。我们发现，在Caco-2细胞体外分化过程中，ST6GalNAc-I转录水平与CDX2表达具有协同性，并且在MKN45胃癌细胞中过表达CDX2可增加ST6GalNAc-I转录水平。通过染色质免疫沉淀技术在Caco-2细胞和IM中鉴定并分析了ST6GalNAc-I调控序列中的9个假定CDX结合位点。结果显示，CDX2蛋白被募集到所有区域，其中最靠近近端的位点在体内优先被占据。荧光素酶检测表明，CDX2能够反式激活ST6GalNac-I调控区域。对于位于ATG起始密码子附近的区域，诱导作用更强，对这些位点进行定点诱变证实了它们的重要性。总之，我们表明CDX2转录调控ST6GalNAc-I基因表达，特别是在癌前IM病变中。
