Wu Hui-Hui, Li Yan-Liang, Liu Nai-Jia, Yang Zhen, Tao Xiao-Ming, Du Yan-Ping, Wang Xuan-Chun, Lu Bin, Zhang Zhao-Yun, Hu Ren-Ming, Wen Jie
Department of Endocrinology and Metabolism, Jing'an District Center Hospital of Shanghai, Shanghai, 200040 China.
2Department of Endocrinology and Metabolism, Huashan Hospital of Fudan University, NO. 12 Wulumuqi Mid Road, Building 0#, Jing'an District, Shanghai, 200040 China.
Diabetol Metab Syndr. 2019 Jul 5;11:55. doi: 10.1186/s13098-019-0449-3. eCollection 2019.
Transcription factor 7-like 2 (TCF7L2), which previously known as TCF-4, is a major form of transcription factor involved in the downstream WNT signaling and exhibits the strongest association to diabetes susceptibility. Although we still do not know mechanistically how TCF7L2 exerts its physiological functions on pancreatic endocrine cells, it had been suggested that TCF7L2 may directly affect β-cell function by regulating the activation of PI3K/AKT signaling pathway.
MIN6 cells were transfected with TCF7L2 knockdown virus or lenti-TCF7L2 virus for 48 h to evaluate the contribution of TCF7L2 to the PI3K/AKT signaling pathway and pancreatic β-cell function. This was confirmed by measuring the expression of PI3K p85 and p-Akt by western blotting and insulin secretion by enzyme-linked immunosorbent assay (ELISA), respectively. Chromatin immunoprecipitation (ChIP) and polymerase chain reaction (PCR) experiments were performed to explore the genomic distribution of TCF7L2-binding sites in the promoter of PIK3R1, the affinity between which was analyzed by the luciferase reporter assay.
In the present study, we strikingly identified that TCF7L2 could profoundly inhibit the expression of PIK3R1 gene and its encoding protein PI3K p85, which then could lead to the activation of PI3K/AKT signaling and stimulate insulin secretion in pancreatic β-cells. However, the integrity and stability of evolutionarily conserved TCF7L2-binding motif plays a very crucial role in the binding events between transcription factor TCF7L2 and its candidate target genes. We also found that the affinity of TCF7L2 to the promoter region of PIK3R1 alters upon the specific binding sites, which further provides statistical validation to the necessity of TCF7L2-binding motif.
This study demonstrated that TCF7L2 is closely bound to the specific binding regions of PIK3R1 promoter and prominently controls the transcription of its encoding protein p85, which further affects the activation of PI3K/AKT signaling pathway and insulin secretion.
转录因子7样蛋白2（TCF7L2），以前称为TCF-4，是参与下游WNT信号传导的主要转录因子形式，与糖尿病易感性的关联最为密切。尽管我们仍不清楚TCF7L2如何在胰腺内分泌细胞上发挥其生理功能，但有人提出TCF7L2可能通过调节PI3K/AKT信号通路的激活直接影响β细胞功能。
将TCF7L2敲低病毒或慢病毒-TCF7L2转染MIN6细胞48小时，以评估TCF7L2对PI3K/AKT信号通路和胰腺β细胞功能的作用。分别通过蛋白质印迹法检测PI3K p85和p-Akt的表达以及通过酶联免疫吸附测定（ELISA）检测胰岛素分泌来证实这一点。进行染色质免疫沉淀（ChIP）和聚合酶链反应（PCR）实验，以探索PIK3R1启动子中TCF7L2结合位点的基因组分布，并通过荧光素酶报告基因测定分析其亲和力。
在本研究中，我们惊人地发现TCF7L2可显著抑制PIK3R1基因及其编码蛋白PI3K p85的表达，进而导致PI3K/AKT信号通路的激活并刺激胰腺β细胞中的胰岛素分泌。然而，进化保守的TCF7L2结合基序的完整性和稳定性在转录因子TCF7L2与其候选靶基因之间的结合事件中起着至关重要的作用。我们还发现，TCF7L2与PIK3R1启动子区域的亲和力会因特定结合位点而改变，这进一步为TCF7L2结合基序的必要性提供了统计学验证。
本研究表明，TCF7L2与PIK3R1启动子的特定结合区域紧密结合，并显著控制其编码蛋白p85的转录，进而影响PI3K/AKT信号通路的激活和胰岛素分泌。
