[Experimental study on protective effect of small interfering RNA-induced Atrogin-1 gene silencing on muscle cell malnutrition].
OBJECTIVE
To investigate the protective effect of Atrogin-1 gene silencing via RNA interference technique on a model of muscle cell malnutrition.


METHODS
Sequences 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.


RESULTS
The 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.


CONCLUSIONS
Atrogin-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.
Yuan
Lei
L
Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai 200032, China.
Wu
Guo-Hao
GH
Zhang
Bo
B
D000818:Animals; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D020868:Gene Silencing; D005822:Genetic Vectors; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D032342:Muscle Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009124:Muscle Proteins / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*; D044843:SKP Cullin F-Box Protein Ligases / Q000235:genetics*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D009124:Muscle Proteins; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; C440907:FBXO32 protein, human; D044843:SKP Cullin F-Box Protein Ligases