Zhao Na, Li Ying-Ying, Xu Jia-Man, Yang Mu-Yao, Li Yun-Zhe, Lam Thomas Chuen, Zhou Lei, Tong Qi-Hu, Zhang Jun-Tao, Wang Sheng-Zhan, Hu Xin-Xin, Wu Yu-Fei, Lu Qin-Kang, Lang Ting-Yuan
Ophthalmology Center, the Affiliated People's Hospital of Ningbo University, Ningbo 315040, Zhejiang Province, China.
Wenzhou Medical University, Wenzhou 325035, Zhejiang Province, China.
Int J Ophthalmol. 2024 Nov 18;17(11):1995-2006. doi: 10.18240/ijo.2024.11.04. eCollection 2024.
To investigate the proliferation regulatory effect of cone-rod homeobox (CRX) in retinal pigment epithelium (RPE) and retinoblastoma (RB) cells to explore the potential application and side effect (oncogenic potential) of CRX-based gene therapy in RPE-based retinopathies.
Adult human retinal pigment epithelial (ARPE)-19 and human retinal pigment epithelial (RPE)-1 cells and Y79 RB cell were used in the study. Genetic manipulation was performed by lentivirus-based technology. The cell proliferation was determined by a CellTiter-Glo Reagent. The mRNA and protein levels were determined by quantitative real-time polymerase chain reaction (qPCR) and Western blot assay. The transcriptional activity of the promoter was determined by luciferase reporter gene assay. The bindings between CRX and transcription factor 7 (TCF7) promoter as well as TCF7 and the promoters of TCF7 target genes were examined by chromatin immunoprecipitation (ChIP) assay. The transcription of the TCF7 was determined by a modified nuclear run-on assay.
CRX overexpression and knockdown significantly increased (=3, <0.05 in all the cells) and decreased (=3, <0.01 in all the cells) the proliferation of RPE and RB cells. CRX overexpression and knockdown significantly increased and deceased the mRNA levels of Wnt signaling target genes [including MYC proto-oncogene (), , FOS like 1 (), , cyclin D2 (), cyclin D3 (), cellular communication network factor 4 (), peroxisome proliferator activated receptor delta (), and matrix metallopeptidase 7 ()] and the luciferase activity driven by the Wnt signaling transcription factor (TCF7). TCF7 overexpression and knockdown significantly increased and decreased the proliferation of RPE and RB cells and depletion of TCF7 significantly abolished the stimulatory effect of CRX on the proliferation of RPE and RB cells. CRX overexpression and knockdown significantly increased and decreased the mRNA level of TCF7 and the promoter of TCF7 was significantly immunoprecipitated by CRX antibody.
CRX transcriptionally activates TCF7 to promote the proliferation of RPE and RB cells . CRX is a potential target for RPE-based regenerative medicine. The potential risk of this strategy, tumorigenic potential, should be considered.
研究视锥-视杆同源盒基因（CRX）对视网膜色素上皮（RPE）细胞和成视网膜细胞瘤（RB）细胞增殖的调控作用，以探索基于CRX的基因治疗在基于RPE的视网膜病变中的潜在应用及副作用（致癌潜能）。
本研究使用成人人类视网膜色素上皮（ARPE）-19细胞、人类视网膜色素上皮（RPE）-1细胞和Y79 RB细胞。采用基于慢病毒的技术进行基因操作。通过CellTiter-Glo试剂测定细胞增殖。通过定量实时聚合酶链反应（qPCR）和蛋白质免疫印迹法检测mRNA和蛋白质水平。通过荧光素酶报告基因测定法测定启动子的转录活性。通过染色质免疫沉淀（ChIP）试验检测CRX与转录因子7（TCF7）启动子以及TCF7与其靶基因启动子之间的结合。通过改良的细胞核转录分析测定TCF7的转录。
CRX过表达和敲低分别显著增加（n = 3，所有细胞中P < 0.05）和降低（n = 3，所有细胞中P < 0.01）RPE细胞和RB细胞的增殖。CRX过表达和敲低分别显著增加和降低Wnt信号靶基因[包括原癌基因MYC（）、 、FOS样蛋白1（）、 、细胞周期蛋白D2（）、细胞周期蛋白D3（）、细胞通讯网络因子4（）、过氧化物酶体增殖物激活受体δ（）和基质金属肽酶7（）]的mRNA水平以及由Wnt信号转录因子（TCF7）驱动的荧光素酶活性。TCF7过表达和敲低分别显著增加和降低RPE细胞和RB细胞的增殖，而TCF7的缺失显著消除了CRX对RPE细胞和RB细胞增殖的刺激作用。CRX过表达和敲低分别显著增加和降低TCF7的mRNA水平，且TCF7启动子能被CRX抗体显著免疫沉淀。
CRX通过转录激活TCF7促进RPE细胞和RB细胞的增殖。CRX是基于RPE的再生医学的一个潜在靶点。应考虑该策略的潜在风险，即致癌潜能。
