GATA6调控胆管癌细胞67kDa层粘素受体过表达的分子机制研究

Cholangiocarcinoma (CCA) has one of the highest mortality rates among solid malignancies because of the early and strong invasiveness of the tumor. Our previous studies showed that 67kDa laminin receptor (67LR) was overexpressed in CCAs and promoted CCA invasion and metastasis through various mechanisms. GATA6, which belongs to a conserved transcriptional family of GATA, is the regulator of 67LR. GATA6 regulated transcriptional activity of 67LR though binding the promotor region directly or recruit other factors.In present study, we try to demontrate the relationship between GATA6 and 67LR in clinical CCA specimens by immunohistochemistry, screen the factors recruited by GATA6 by protein array,and then study the mechanism of GATA6 regulating 67LR transcriptional activity using luciferase reporter gene assay, deletion mutations, point mutations, electrophoretic mobility shift and chromatin immunoprecipitation assay. The aim of this study is to enrich the pathogenesis of CCA metastasis and also to procide more potential research evidence for clinical therapy of CCA.

67kDa层粘素受体（67LR）过表达是多数实体恶性肿瘤的共同现象，我们前期研究表明，胆管癌中67LR过表达，通过多种机制促进侵袭转移。GATA6是进化保守的转录因子，胆管癌细胞中GATA6在转录水平调控67LR表达，但确切的调控机制还不清楚。文献报道GATA6可直接与启动子中GATA位点结合，也可能通过募集其他转录因子形成复合体后调节基因转录活性。本研究拟收集临床胆管癌标本进一步验证GATA6与67LR表达相关性，拟用荧光素酶报告基因实验、启动子缺失突变、点突变、电泳迁移率及染色质免疫共沉淀实验研究GATA6与GATA位点结合对67LR转录活性的影响；采用转录因子芯片联合蛋白质免疫共沉淀技术筛选能与GATA6募集的转录因子；通过点突变、染色质免疫共沉淀实验进一步研究复合体与启动子的结合位点；旨在阐明GATA6调控67LR的分子机制，寻找关键的分子靶点，为临床治疗方法的探索提供新思路。

我们前期研究表明，胆管癌中67kDa层粘素受体（67LR）过表达，通过多种机制促进侵袭转移。然而67LR过表达机制不明。GATA6是进化保守的转录因子，其在多种肿瘤中异常表达，在侵袭转移中发挥重要作用。我们前期预实验发现胆管癌细胞中GATA6在转录水平调控67LR表达，但确切的调控机制还不清楚。本研究通过临床胆管癌标本及胆管癌细胞体外培养，采用免疫组化、PCR、WB、荧光素酶报告基因实验、ChIP、ChIP-sequence芯片等研究，发现：（1）胆管癌细胞中GATA6异常表达，与侵袭转移特性显著相关，与预后不良显著相关；（2）胆管癌细胞中GATA6通过上调67LR促进侵袭转移；（3）胆管癌细胞中，GATA6在转录水平调控67LR，其直接与67LR启动子区-498位点和-681位点结合，增强其转录活性，上调67LR表达；（4）层粘素-5特异性γ2链在胆管癌细胞中表达上调，显著促进侵袭转移，提示层粘素-5是67LR发挥作用的重要配体；（5）胆管癌细胞中GATA6与LOXL2结合，共同调控67LR表达。本研究阐明了胆管癌中GATA6调控67LR的分子机制，托看了胆管癌侵袭转移机制研究，并为胆管癌临床抗侵袭转移靶向治疗提供了实验依据。