PPM1D silencing by lentiviralmediated RNA interfe

Authors: Peng Wang Jing Rao Haifeng Yang Hongyang Zhao Lin Yang

Publish Date: 2011/02/19

Volume: 31, Issue: 1, Pages: 94-99

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Abstract

To construct a lentiviral shRNA vector targeting human protein phosphatase 1D magnesiumdependent PPM1D gene and detect its effectiveness of gene silencing in human gliomas specific siRNA targets with short hairpin frame were designed and synthesized DNA oligo was cloned into the pFUGWiRNA lentiviral expression vector and then PCR and sequencing analyses were conducted to verify the constructs After the verified plasmids were transfected into 293T cells the lentivirus was produced and the titer of virus was determined Realtime quantitative PCR and Western blot were performed to detect the PPM1D expression level in the infected glioma cells PCR and Western blot analyses revealed the optimal interfering target and the virus with a titer of 6×108 TU/mL was successfully packaged The PPM1D expression in human glioma cells was knocked down at both mRNA and protein levels by virus infection The expression of PPM1D mRNA and protein was decreased by 763 and 870 respectively as compared with control group The multiple functions of human glioma cells after PPM1D RNA interference were detected by flow cytometry and cell counting kit8 CCK8 Efficient downregulation of PPM1D resulted in significantly increased cell apoptosis and reduced cell proliferation and invasion potential in U87MG cells We have successfully constructed the lentiviral shRNA expression vector capable of stable PPM1D gene silencing at both mRNA and protein levels in glioma cells And our data gave evidence that the reduced cell growth observed after PPM1D silencing in glioma cells was at least partly due to increased apoptotic cell death

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