ShRNA-mediated silencing of MDM2 inhibits growth of HepG2 hepatocellular carcinoma cells xenografted in nude mice.
- Author:
Yan-ying ZHAO
1
;
Ya-gang LI
;
Yuan-jie SUN
;
Hai-peng LIU
;
Ze-cheng YANG
;
Duo-duo ZHANG
;
Chun-yan ZHAO
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Carcinoma, Hepatocellular; genetics; pathology; Cell Proliferation; Hep G2 Cells; Humans; Liver Neoplasms; genetics; pathology; Male; Mice; Mice, Nude; Plasmids; Proto-Oncogene Proteins c-mdm2; genetics; metabolism; RNA Interference; RNA, Messenger; genetics; RNA, Small Interfering; Transfection; Tumor Suppressor Protein p53; metabolism; Xenograft Model Antitumor Assays
- From: Chinese Journal of Hepatology 2013;21(3):213-217
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct a short hairpin (sh)RNA targeting the gene encoding the MDM2 oncoprotein in order to investigate its role in human hepatocellular carcinoma (HCC) and its potential for use as a gene therapy strategy to inhibit HCC growth in vivo.
METHODSSmall interfering (si)RNAs were designed targeting the MDM2 gene (siMDM2-1 and siMDM2-2) and unrelated sequences (negative control) and cloned into the expression plasmid pGCSilencer-U6-neo-GFP. A HCC mouse model was established by subcutaneous inoculation of HepG2 cells (2 x 10(6) in 0.2 ml) into 20 nude mice. The inoculated mice were divided into four equal groups for tumor-localized injections of saline, negative control siRNA plasmid, siMDM2-1 plasmid, and siMDM2-2 plasmid. Tumor growth was observed daily (by caliper measurement) for one month, when mice were sacrificed by cervical dislocation. The tumor mass was resected for analysis of tumor inhibition rate (% = [(average tumor weight of control group - average tumor weight of treatment group) / average tumor weight of control group x 100]) and effects on MDM2 and p53 mRNA and protein expression (by reverse transcription- PCR and western blotting, both normalized to beta-actin). Significance of between-group differences was assessed by one-way ANOVA or LSD test; pairwise comparisons were made by the Chi-squared test.
RESULTSsiMDM2-1 and siMDM2-2 suppressed the xenografted tumor growth remarkably (60.6% and 54.6% inhibition rates, respectively), significantly reduced the expression ofMDM2 gene (62.8% and 61.6%) and protein (60.7% and 59.5%), and significantly increased p53 gene (47.1% and 45.6%) and protein (45.9% and 44.3%) (all, P < 0.05).
CONCLUSIONshRNA-mediated silencing of the MDM2 gene effectively inhibits HCC tumorigenesis of subcutaneously xenografted HepG2 cells in nude mice, and the mechanism may involve p53.