Effects Of The Human21.5kD MBP Gene Silencing On The Proliferation And Apoptosis Of Glioma Cells

Objective: To investigate the effects of silencing of the human cerebral21.5kD myelin basic protein (MBP) gene on the proliferation and apoptosis ofthe glioma U251cells.Methods:(1) Three short hair-pin RNA (shRNA) recombinantplasmids(pGenesil-1-MBP-1,pGenesil-1-MBP-2,pGenesil-1-MBP-3and onenegative control plasmid pGenesil-1-MBP-neg) against the21.5kD MBP genewere constructed with application of molecular cloning techniques, and theplasmids were then screened using restriction enzyme analysis and sequenceanalysis.(2) The four recombinant plasmids were transfected into the humanglioma cell line U251via a liposome-mediated route, the transfection rateswere observed24h after transfection, and the amounts of the mRNAexpression in the U251cells were measured using real-time quantitative PCRto reflect the silencing effect of recombinant plasmids.(3) Theplasmid-transfected group with the best silencing effect was used as anexperimental group, the group transfected with negative control plasmids usedas a negative control group, and the group transfected with lisosomes used asa blank control group. In these three groups, the levels of the MBP proteinexpression in the U251cells were measured with Westernblot, and the effectsof MBP on the cellular proliferation and apoptosis were detected using theCCK-8cell proliferation assay and the apoptosis detection agent AnnexinV-PE, respectively.Results:(1) The four shRNA recombinant plasmids were successfullyconstructed.(2) The four recombinant plasmids were successfully transfected into the glioma cell line U251, with a transfection rate of80%. ThepGenesil-1-MBP-3transfected group, with the lowest relative amount ofmRNA expression, was the best silencing plasmid group (experimental group).(3) The MBP level or cellular proliferation activity in the experimental groupwas significantly lower than those in the negative control and blank controlgroups (P<0.05); the cellular apoptotic rate in the experimental group wassignificantly higher than those in the other2groups (P<0.05).Conclusion: High expression of the human cerebral21.5kD MBP gene,may be closely related to the occurrence and development ofglioma.;silencing of the human cerebral21.5kD MBP gene played dual rolesin the inhibition of proliferation and the promotion of apoptosis of the gliomaU251cells.