title: PPM1D silencing by lentiviral-mediated RNA interference inhibits proliferation and invasion of human glioma cells.
abstract: To construct a lentiviral shRNA vector targeting human protein phosphatase 1D magnesium-dependent (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 pFU-GW-iRNA 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. Real-time 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 6×10(8) 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 76.3% and 87.0% 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 kit-8 (CCK-8). Efficient down-regulation of PPM1D resulted in significantly increased cell apoptosis and reduced cell proliferation and invasion potential in U87-MG 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.
authors.affiliation: Department of Neurosurgery, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, 510080, China.|Department of Neurosurgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Wang
authors.affiliation: Department of Neurosurgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Rao
authors.affiliation: Department of Neurosurgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
authors.forename: Haifeng
authors.initials: H
authors.lastname: Yang
authors.affiliation: Department of Neurosurgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
authors.forename: Hongyang
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Department of Neurosurgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China. linyang@hust.edu.cn.
authors.forename: Lin
authors.initials: L
authors.lastname: Yang
mesh_terms: D017209:Apoptosis / Q000235:genetics*; D001932:Brain Neoplasms / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D055785:Gene Knockdown Techniques; D005910:Glioma / Q000235:genetics* / Q000473:pathology; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism; D009361:Neoplasm Invasiveness / Q000517:prevention & control; D010749:Phosphoprotein Phosphatases / Q000235:genetics* / Q000378:metabolism; D000071636:Protein Phosphatase 2C; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; C000607458:PPM1D protein, human; D010749:Phosphoprotein Phosphatases; D000071636:Protein Phosphatase 2C