Moazzeni Hamidreza, Akbari Mohammad Taghi, Yazdani Shahin, Elahi Elahe
Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box. 14115-331, Tehran, Iran.
Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box. 14115-331, Tehran, Iran.
Gene. 2016 Nov 15;593(1):76-83. doi: 10.1016/j.gene.2016.08.019. Epub 2016 Aug 9.
The transcription factor PITX2 is implicated in glaucoma pathology. In an earlier study we had used microarray analysis to identify genes in the trabecular meshwork (TM) that are affected by knock down of PITX2. Here, those studies were pursued to identify genes that are direct targets of PITX2 and that may be relevant to glaucoma. Initially, bioinformatics tools were used to select among the genes that had been affected by PITX2 knock down those that have PITX2 binding sites and that may be involved in glaucoma related functions. Subsequently, the effect of PITX2 was tested using the dual luciferase assay in four cell cultures including two primary TM cultures co-transfected with vectors containing promoter fragments of six candidate genes upstream of a luciferase gene and a vector that expressed PITX2. Finally, the effect of PITX2 on endogenous expression of two genes was assessed by over expression and knock down of PITX2 in TM cells. Thirty four genes were found to contain PITX2 binding sites in their putative promoter regions, and 16 were found to be associated with TM-specific and/or glaucoma associated functions. Results of dual luciferase assays confirmed that two of six genes tested were directly targeted by PITX2. The two genes were CXCL6 (chemokine (C-X-C motif) ligand 6) and BBS5 (Bardet-Biedl syndrome 5). Over expression and knock down of PITX2 showed that this transcription factor affects endogenous expression of these two genes in TM cells. CXCL6 encodes a pro-inflammatory cytokine, and many studies have suggested that cytokines and other immune system functions are involved in glaucoma pathogenesis. BBS5 is a member of the BBS family of genes that affect ciliary functions, and ciliary bodies in the anterior chamber of the eye produce the aqueous fluid that affects intraocular pressure. Immune related functions and intraocular pressure are both important components of glaucoma pathology. The role of PITX2 in glaucoma may be mediated partly by regulating the expression of CXCL6 and BBS5 and thus affecting immune functions and intraocular pressure.
转录因子PITX2与青光眼病理相关。在早期研究中，我们使用微阵列分析来鉴定小梁网（TM）中受PITX2敲低影响的基因。在此，继续开展这些研究以鉴定作为PITX2直接靶标的、可能与青光眼相关的基因。最初，利用生物信息学工具从受PITX2敲低影响的基因中筛选出那些具有PITX2结合位点且可能参与青光眼相关功能的基因。随后，在四种细胞培养物中使用双荧光素酶测定法检测PITX2的作用，其中包括两种原代TM培养物，它们与含有荧光素酶基因上游六个候选基因启动子片段的载体以及表达PITX2的载体共转染。最后，通过在TM细胞中过表达和敲低PITX2来评估其对两个基因内源性表达的影响。发现34个基因在其假定的启动子区域含有PITX2结合位点，16个基因被发现与TM特异性和/或青光眼相关功能有关。双荧光素酶测定结果证实，所检测的六个基因中有两个是PITX2的直接靶标。这两个基因是CXCL6（趋化因子（C-X-C基序）配体6）和BBS5（巴德-比埃尔综合征5）。PITX2的过表达和敲低表明，该转录因子影响TM细胞中这两个基因的内源性表达。CXCL6编码一种促炎细胞因子，许多研究表明细胞因子和其他免疫系统功能参与青光眼发病机制。BBS5是影响纤毛功能的BBS基因家族成员，眼房水中的睫状体产生影响眼压的房水。免疫相关功能和眼压都是青光眼病理的重要组成部分。PITX2在青光眼中的作用可能部分是通过调节CXCL6和BBS 的表达，从而影响免疫功能和眼压来介导的。
