Department of Molecular Biology, Institute for Biological Research "Siniša Stanković" - National Institute of Republic of Serbia, University of Belgrade, Belgrade, Serbia.
Institute of Biochemistry and Technical Biochemistry, University of Stuttgart, Stuttgart, Germany.
Front Endocrinol (Lausanne). 2023 Mar 16;14:1134478. doi: 10.3389/fendo.2023.1134478. eCollection 2023.
Beta cell dysfunction by loss of beta cell identity, dedifferentiation, and the presence of polyhormonal cells are main characteristics of diabetes. The straightforward strategy for curing diabetes implies reestablishment of pancreatic beta cell function by beta cell replacement therapy. Aristaless-related homeobox (Arx) gene encodes protein which plays an important role in the development of pancreatic alpha cells and is a main target for changing alpha cell identity.
In this study we used CRISPR/dCas9-based epigenetic tools for targeted hypermethylation of Arx gene promoter and its subsequent suppression in mouse pancreatic αTC1-6 cell line. Bisulfite sequencing and methylation profiling revealed that the dCas9-Dnmt3a3L-KRAB single chain fusion constructs (EpiCRISPR) was the most efficient. Epigenetic silencing of expression was accompanied by an increase in transcription of the insulin gene () mRNA on 5 and 7 post-transfection day, quantified by both RT-qPCR and RNA-seq. Insulin production and secretion was determined by immunocytochemistry and ELISA assay, respectively. Eventually, we were able to induce switch of approximately 1% of transiently transfected cells which were able to produce 35% more insulin than Mock transfected alpha cells.
In conclusion, we successfully triggered a direct, transient switch of pancreatic alpha to insulin-producing cells opening a future research on promising therapeutic avenue for diabetes management.
β细胞功能障碍的特征包括β细胞丧失身份、去分化和多激素细胞的存在，这些都是糖尿病的主要特征。治疗糖尿病的直接策略意味着通过β细胞替代疗法重建胰腺β细胞功能。Aristaless 相关同源盒（Arx）基因编码的蛋白在胰腺α细胞的发育中起着重要作用，是改变α细胞身份的主要靶点。
在这项研究中，我们使用基于 CRISPR/dCas9 的表观遗传学工具对 Arx 基因启动子进行靶向超甲基化，并随后在小鼠胰腺αTC1-6 细胞系中抑制其表达。亚硫酸氢盐测序和甲基化分析显示，dCas9-Dnmt3a3L-KRAB 单链融合构建物（EpiCRISPR）最为有效。 表达的表观遗传沉默伴随着胰岛素基因（）mRNA 的转录增加，在转染后第 5 天和第 7 天通过 RT-qPCR 和 RNA-seq 进行定量。通过免疫细胞化学和 ELISA 测定分别确定胰岛素的产生和分泌。最终，我们能够诱导大约 1%的瞬时转染细胞发生开关，这些细胞产生的胰岛素比 Mock 转染的α细胞多 35%。
总之，我们成功地触发了胰腺α细胞向胰岛素分泌细胞的直接、瞬时转换，为糖尿病治疗开辟了有前途的研究途径。
