抑制结缔组织生长因子过表达可减少肝癌细胞在体外和体内的生长。

Inhibition of connective tissue growth factor overexpression decreases growth of hepatocellular carcinoma cells in vitro and in vivo.

机构信息

Department of Pathology, School of Medicine, Yangzhou University, Yangzhou, Jiangsu 225500, China.

出版信息

Chin Med J (Engl). 2011 Nov;124(22):3794-9.

Abstract

BACKGROUND

We have previously found that connective tissue growth factor (CTGF) is highly expressed in a rat model of liver cancer. Here, we examined expression of CTGF in human hepatocellular carcinoma (HCC) cells and its effect on cell growth.

METHODS

Real-time PCR was used to observe expression of CTGF in human HCC cell lines HepG2, SMMC-7721, MHCC-97H and LO2. siRNA for the CTGF gene was designed, synthesized and cloned into a Plk0.1-GFP-SP6 vector to construct a lentivirus-mediated shRNA/CTGF. CTGF mRNA and protein expression in HepG2 cells treated by CTGF-specific shRNA was evaluated by real-time PCR and Western blotting. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was utilized to evaluate the growth effect, and a colony formation assay was used for observing clonogenic growth. In vivo, tumor cell proliferation was evaluated in a nude mouse model of xenotransplantation. Statistical significance was determined by t test for comparison between two groups, or analysis of variance (ANOVA) for multiple groups.

RESULTS

Immunohistochemical staining of CTGF was seen in 35 of 40 HCC samples (87.5%). CTGF was overexpressed 5-fold in 20 HCC tissues, compared with surrounding non-tumor liver tissue. CTGF mRNA level was 5 - 8-fold higher in HepG2, SMMC-7721 and MHCC-97H than in LO2 cells. This indicated that the inhibition rate of cell growth was 43% after knockdown of CTGF expression (P < 0.05). Soft agar colony formation assay showed that siRNA mediated knockdown of CTGF inhibited colony formation in soft agar of HepG2 cells (P < 0.05). The volume of tumors from CTGF-shRNA-expressing cells only accounted for 35% of the tumors from the scrambled control-infected HepG2 cells (P < 0.05).

CONCLUSIONS

CTGF was overexpressed in human HCC cells and downregulation of CTGF inhibited HCC growth in vitro and in vivo. Knockdown of CTGF may be a potential therapeutic strategy for treatment of HCC.

摘要

背景

我们之前发现结缔组织生长因子（CTGF）在大鼠肝癌模型中高表达。在这里，我们检测了 CTGF 在人肝癌细胞（HCC）系 HepG2、SMMC-7721、MHCC-97H 和 LO2 中的表达，并观察其对细胞生长的影响。

方法

采用实时 PCR 观察 CTGF 在 HepG2、SMMC-7721、MHCC-97H 和 LO2 人肝癌细胞系中的表达。设计、合成 CTGF 基因的 siRNA，并克隆到 Plk0.1-GFP-SP6 载体中，构建慢病毒介导的 shRNA/CTGF。用实时 PCR 和 Western blot 检测 CTGF 特异性 shRNA 处理 HepG2 细胞后 CTGF mRNA 和蛋白的表达。3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法评估生长效应，集落形成实验观察克隆生长。体内，在裸鼠异种移植模型中评估肿瘤细胞增殖。两组间比较采用 t 检验，多组间比较采用方差分析（ANOVA）。

结果

40 例 HCC 标本中有 35 例（87.5%）CTGF 免疫组化染色阳性。20 例 HCC 组织中 CTGF 表达上调 5 倍，高于周围非肿瘤肝组织。HepG2、SMMC-7721 和 MHCC-97H 细胞 CTGF mRNA 水平比 LO2 细胞高 5-8 倍。提示 CTGF 表达下调后细胞生长抑制率为 43%（P < 0.05）。软琼脂集落形成实验显示，siRNA 介导的 CTGF 下调抑制 HepG2 细胞软琼脂集落形成（P < 0.05）。CTGF-shRNA 表达细胞的肿瘤体积仅占 scrambled 对照感染 HepG2 细胞肿瘤体积的 35%（P < 0.05）。