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Molecular Modulation by Lentivirus-Delivered Specific shRNAs in Endoplasmic Reticulum Stressed Neurons
Published on: April 24, 2021
Mpz gene suppression by shRNA increases Schwann cell apoptosis in vitro
Li Lei1, Demin Han, Shusheng Gong
1Department of Otolaryngology, Affiliated Beijing Tongren Hospital, Capital Medical University, 1 Dongjiaominxiang, Dongcheng District, Beijing, 100730, People's Republic of China.
Abstract:
We investigated the effects of short hairpin RNA (shRNA) on myelin protein zero (MPZ) gene expression in Schwann cells (SCs) in vitro and determined the effects of the MPZ gene suppression on the survival of SCs. The MPZ-specific shRNA was introduced into a lentiviral vector for expression under the U6 promoter, and the viral vector-based shRNAs were used to infect cultured SCs. The efficiency of MPZ knockdown was analyzed by real time-PCR (RT-PCR) and western blotting. Flow cytometric analysis and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were used to determine the cell cycle and the amount of apoptosis of SCs. We found that MPZ shRNAs significantly inhibited the expression of the MPZ gene and induced SC apoptosis in vitro. These results provided interesting experimental evidence for understanding the mechanism of demyelinating neuropathies caused by MPZ gene malfunction.
Insights
Short hairpin RNA (shRNA) targeting the myelin protein zero (MPZ) gene was used to suppress MPZ expression in Schwann cells (SCs). This suppression led to increased SC apoptosis, offering insights into demyelinating neuropathies.
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Biology
Background:
- Myelin protein zero (MPZ) is crucial for myelin sheath formation and maintenance in the peripheral nervous system.
- Dysfunction of the MPZ gene is implicated in various demyelinating neuropathies.
- Schwann cells (SCs) are the primary glial cells responsible for myelination in the PNS.
Purpose of the Study:
- To investigate the impact of short hairpin RNA (shRNA)-mediated MPZ gene suppression on SCs in vitro.
- To determine the effect of reduced MPZ expression on SC survival and apoptosis.
Main Methods:
- MPZ-specific shRNA was delivered into cultured SCs using a lentiviral vector.
- MPZ gene knockdown efficiency was quantified using real-time PCR (RT-PCR) and western blotting.
- SC cell cycle progression and apoptosis rates were assessed via flow cytometry and TUNEL assays.
Main Results:
- MPZ-specific shRNAs effectively inhibited MPZ gene expression in SCs.
- Suppression of MPZ gene expression resulted in a significant increase in SC apoptosis.
- Inhibition of MPZ expression altered SC cell cycle dynamics.
Conclusions:
- Targeted MPZ gene suppression using shRNA induces apoptosis in Schwann cells.
- These findings provide experimental evidence for the role of MPZ in SC survival.
- Understanding MPZ's function is critical for elucidating mechanisms of MPZ-related demyelinating neuropathies.
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