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17 February 2019 : Laboratory Research  

Phosphatase Actin Regulator-1 (PHACTR-1) Knockdown Suppresses Cell Proliferation and Migration and Promotes Cell Apoptosis in the bEnd.3 Mouse Brain Capillary Endothelial Cell Line

Yao Jing1BCDE, Lin Zhang1BC, Zhiming Xu1CDE, Hao Chen1AB, Shiming Ju1ABC, Jun Ding1ABEFG, Yan Guo1CDE*, Hengli Tian1AFG

DOI: 10.12659/MSM.912586

Med Sci Monit 2019; 25:1291-1300

Abstract

BACKGROUND: The phosphatase actin regulator-1 (PHACTR-1) gene on chromosome 6 encodes an actin and protein phosphatase 1 (PP1) binding protein, Phactr-1, which is highly expressed in brain tissues. Phactr-1 expression is involved in physiological and pathological cerebral microvascular events. This study aimed to investigate the role of expression of Phactr-1 in a mouse brain capillary endothelial cell line, bEnd.3, by knockdown the PHACTR-1 gene.

MATERIAL AND METHODS: Three bEnd.3 cell groups were studied, CON (normal control cells), NC (control scramble transfected cells), and KD (cells with PHACTR-1 gene knockdown). The PHACTR-1 gene was knocked down using transfection with small hairpin RNA (shRNA). In the three cell groups cell proliferation, migration, and apoptosis were studied by MTT and colony formation assays, transwell and scratch assays, and flow cytometry. The related cell pathways of associated with Phactr-1 knockdown were studied by Western blot.

RESULTS: Phactr-1 knockdown suppressed bEnd.3 cell proliferation and migration, promoted cell apoptosis, and downregulated the expressions of migration-associated proteins, including matrix metalloproteinase (MMP)-2 and MMP-9 and upregulated apoptosis-associated proteins, including Bax, Bcl-2, cleaved caspase-3, and caspase-3.

CONCLUSIONS: Phactr-1 was shown to have a role in the inhibition of endothelial cell proliferation and migration, promoted cell apoptosis, and regulated matrix metalloproteinases and apoptosis-associated proteins. These findings indicate that the expression of the Phactr-1 should be studied further in the cerebral microvasculature, both in vitro and in vivo, regarding its potential as a diagnostic and therapeutic target for cerebral microvascular disease.

Keywords: Acid Phosphatase, Actins, Apoptosis, Cell Migration Assays, Cell Proliferation, endothelial cells, Brain, Cell Line, Cell Movement, Down-Regulation, Gene Knockdown Techniques, Microfilament Proteins, Phosphoric Monoester Hydrolases, Signal Transduction, Transfection

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Medical Science Monitor eISSN: 1643-3750
Medical Science Monitor eISSN: 1643-3750