Lin Chunjing, Huang Ziyang, Zhou Riyong, Zhou Ying, Shentu Yangping, Yu Kang, Zhang Yu
Department of Gastroenterology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, P.R. China.
Department of Hematology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, P.R. China.
Exp Ther Med. 2021 Feb;21(2):117. doi: 10.3892/etm.2020.9549. Epub 2020 Dec 3.
Notch3 is a member of the Notch family and its mutations are known to cause a hereditary human disorder called cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). However, the specific function and signaling cascade initiated by CADASIL mutants remain unknown. To gain further insight into mechanism of action of CADASIL mutants, the present study conducted several experiments on the effects of Notch3 mutants in multiple cell lines. The protein levels of Notch3, fibronectin, collagen, inducible nitric oxide synthase and DNA (cytosine-5)-methyltransferase 1 (DNMT1) were determined by western blotting. The mRNA levels of IL-1β and TNF-α were measured by reverse transcription semi-quantitative PCR and DNMT1 mRNA levels were determined by quantitative PCR. Trypan blue staining was used for proliferation analysis and wound healing assays were performed to determine cell migration capability. The present study reported that R90C and R169C Notch3 mutants, and wild-type Notch3 had different effects on several cell lines. In T/GHA-VSMC cells, following the transfection of the two mutants, collagen and fibronectin expression increased, whereas expression decreased in IMR-90 cells. In BV2 cells, the two mutants resulted in decreased nitric oxide and iNOS production. In HeLa cells, proliferation and migration increased significantly following the transfection of the two mutants, whereas in the MCF-7 and HCC1937 cell lines, cell proliferation and migration decreased. In addition, the two mutants suppressed the expression of DNMT1 in HeLa and IMR-90 cells. Overall, the present study provided novel insights that further explored the underlying mechanisms of CADASIL.
Notch3是Notch家族的一员，已知其突变会导致一种遗传性人类疾病，称为伴有皮质下梗死和白质脑病的常染色体显性遗传性脑动脉病（CADASIL）。然而，CADASIL突变体引发的具体功能和信号级联反应仍不清楚。为了进一步深入了解CADASIL突变体的作用机制，本研究对Notch3突变体在多种细胞系中的作用进行了多项实验。通过蛋白质印迹法测定Notch3、纤连蛋白、胶原蛋白、诱导型一氧化氮合酶和DNA（胞嘧啶-5）-甲基转移酶1（DNMT1）的蛋白质水平。通过逆转录半定量PCR测量IL-1β和TNF-α的mRNA水平，并通过定量PCR测定DNMT1的mRNA水平。台盼蓝染色用于增殖分析，并进行伤口愈合试验以确定细胞迁移能力。本研究报告称，R90C和R169C Notch3突变体与野生型Notch3对几种细胞系有不同影响。在T/GHA-VSMC细胞中，转染这两种突变体后，胶原蛋白和纤连蛋白表达增加，而在IMR-90细胞中表达降低。在BV2细胞中，这两种突变体导致一氧化氮和诱导型一氧化氮合酶产生减少。在HeLa细胞中，转染这两种突变体后增殖和迁移显著增加，而在MCF-7和HCC1937细胞系中，细胞增殖和迁移减少。此外，这两种突变体抑制了HeLa和IMR-90细胞中DNMT1的表达。总体而言，本研究提供了新的见解，进一步探索了CADASIL的潜在机制。
