Zhao Yan-ying, Li Ya-gang, Sun Yuan-jie, Liu Hai-peng, Yang Ze-cheng, Zhang Duo-duo, Zhao Chun-yan
Department of Digestive System, the Fourth Hospital of Jilin University, Changchun, China.
Zhonghua Gan Zang Bing Za Zhi. 2013 Mar;21(3):213-7.
To 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.
Small 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.
siMDM2-1 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).
shRNA-mediated silencing of the MDM2 gene effectively inhibits HCC tumorigenesis of subcutaneously xenografted HepG2 cells in nude mice, and the mechanism may involve p53.
构建针对编码MDM2癌蛋白基因的短发夹（sh）RNA，以研究其在人肝细胞癌（HCC）中的作用及其作为体内抑制HCC生长的基因治疗策略的潜力。
设计靶向MDM2基因的小干扰（si）RNA（siMDM2-1和siMDM2-2）以及无关序列（阴性对照），并克隆到表达质粒pGCSilencer-U6-neo-GFP中。通过将HepG2细胞（2×10⁶个细胞于0.2 ml中）皮下接种到20只裸鼠中建立HCC小鼠模型。将接种后的小鼠分成四组，每组数量相等，分别进行肿瘤局部注射生理盐水、阴性对照siRNA质粒、siMDM2-1质粒和siMDM2-2质粒。每天（用卡尺测量）观察肿瘤生长情况，持续一个月，然后通过颈椎脱臼法处死小鼠。切除肿瘤块，分析肿瘤抑制率（% = [(对照组平均肿瘤重量 - 治疗组平均肿瘤重量) / 对照组平均肿瘤重量×100]）以及对MDM2和p53 mRNA和蛋白表达的影响（通过逆转录PCR和蛋白质印迹法，两者均以β-肌动蛋白为内参进行标准化）。组间差异的显著性通过单因素方差分析或LSD检验进行评估；两两比较采用卡方检验。
siMDM2-1和siMDM2-2显著抑制异种移植肿瘤的生长（抑制率分别为60.6%和54.6%），显著降低MDM2基因（62.8%和61.6%）和蛋白（60.7%和59.5%）的表达，并显著增加p53基因（47.1%和45.6%）和蛋白（45.9%和44.3%）（所有P < 0.05）。
shRNA介导的MDM2基因沉默有效抑制裸鼠皮下异种移植HepG2细胞的HCC肿瘤发生，其机制可能涉及p53。
