Experimental study on protective effect of small interfering RNA-induced Atrogin-1 gene silencing on muscle cell malnutrition.
- Author:
Lei YUAN
1
;
Guo-Hao WU
;
Bo ZHANG
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Cell Differentiation; Cell Proliferation; Cells, Cultured; Gene Silencing; Genetic Vectors; Lentivirus; genetics; Mice; Muscle Cells; cytology; drug effects; metabolism; Muscle Proteins; genetics; RNA, Small Interfering; genetics; SKP Cullin F-Box Protein Ligases; genetics; Transfection; Tumor Necrosis Factor-alpha; pharmacology
- From: Chinese Journal of Surgery 2009;47(9):705-708
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo investigate the protective effect of Atrogin-1 gene silencing via RNA interference technique on a model of muscle cell malnutrition.
METHODSSequences of five target Atrogin-1 siRNA and the control were selected and synthesized and cloned to vector pBS-hU6-I and then to vector FG12. The length and rightness of the sequences were confirmed. The recombinant FG12 vectors were cotransfected along with pRSVREV, pMDLg/pRRE and pHCMV-G into 293T cells to package lentivirus particles, with which C2C12 cells were infected. The infected C2C12 cells were cultured and differentiated to form myotubes before TNF-alpha was added to induce malnutrition. Expressed products of Atrogin-1 of myotubes were identified by real time PCR and Western blot methods. Myotubes were observed and photographed directly in culture plate without fixation.
RESULTSThe length and sequences of inserted DNA were right. Compared with the RNA interferencing group, significant atrophy and upregulated expression of Atrogin-1 of myotubes treated by TNF-alpha was found in the control group.
CONCLUSIONAtrogin-1 gene silencing could be used to inhibit malnutrition of muscle cells caused by TNF-alpha. Atrogin-1 could be an ideal target in the treatment of cancer cachexia.
