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Study on the mechanism of excessive apoptosis of nucleus pulposus cells induced by shRNA-Piezo1 under abnormal mechanical stretch stress.

Abstract：

OBJECTIVE:The aim of the study was to explore the mechanism of excessive apoptosis of nucleus pulposus cells induced by short hairpin RNA (shRNA) Piezo type mechanosensitive ion channel component 1 (Piezo1) under abnormal mechanical stretch stress. METHODS:In vitro mechanical stretch stress model of nucleus pulposus cells in vitro was established, in which the expression of Piezo1 was interfered by transfection of shRNA-Piezo1 interfering vector. Both messenger RNA and protein level of Piezo1 were measured by reverse-transcription polymerase chain reaction and Western blot analysis, respectively. Cytoplasmic Ca2+ was detected by Fluo3-AM kit, and changes of mitochondrial membrane potential in cells were detected using Cell Meter Assay kit. Finally, the apoptosis was evaluated with annexin V-fluorescein isothiocyanate kit. RESULTS:The highest transfection efficiency of lentivirus titer was 1 × 10 TU/mL and the nucleus pulposus cells were transfected with plural multiplicity of infection = 50. Homo-3201 sequence exhibited the most effective silencing effect and was used in subsequent experiments as the default sequence of shRNA-Piezo1. The calcium content in the cytoplasm of the tension stress group increased significantly compared with that in the blank control group ( q = 3.773; P < 0.05). The level of cytosolic calcium in shRNA-interference group was significantly lower than that in stretch stress group ( q = 5.159; P < 0.05). Stretch stress treatment resulted in an elevated ratio of mitochondrial membrane potential turnover as opposed to blank control group ( q = 4.332; P < 0.05), while shRNA-interference group showed smaller ratio of mitochondrial membrane potential turnover than that in stretch stress group ( q = 4.974; P < 0.05). Similar results were also observed in apoptosis rate analysis ( q = 3.175; P < 0.05). CONCLUSION:ShRNA-Piezo1 can protect cells by reducing the level of intracellular Ca2+ and the change of mitochondrial membrane potential.

journal_name

J Cell Biochem

journal_title

Journal of cellular biochemistry

authors

Yang Q,Zhou Y,Wang J,Fu W,Li X

doi

10.1002/jcb.27683

subject

Has Abstract

pub_date

2019-03-01 00:00:00

pages

3989-3997

issue

eissn

0730-2312

issn

1097-4644

journal_volume

120

pub_type

杂志文章

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Study on the mechanism of excessive apoptosis of nucleus pulposus cells induced by shRNA-Piezo1 under abnormal mechanical stretch stress.