一种针对骨桥蛋白的可诱导短发夹RNA载体可降低人食管鳞状细胞癌在体外和体内的转移潜能。

An inducible short-hairpin RNA vector against osteopontin reduces metastatic potential of human esophageal squamous cell carcinoma in vitro and in vivo.

作者信息

Ito Tetsuo, Hashimoto Yosuke, Tanaka Eiji, Kan Takatsugu, Tsunoda Shigeru, Sato Fumiaki, Higashiyama Motoshige, Okumura Tomoyuki, Shimada Yutaka

机构信息

Department of Surgery and Surgical Basic Science, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

出版信息

Clin Cancer Res. 2006 Feb 15;12(4):1308-16. doi: 10.1158/1078-0432.CCR-05-1611.

DOI:10.1158/1078-0432.CCR-05-1611

PMID:16489088

Abstract

PURPOSE

To elucidate the clinical significance of osteopontin and the effect of conditional down-regulation of osteopontin in esophageal squamous cell carcinoma (ESCC), we investigated osteopontin expression in tumors and tested an inducible osteopontin-short-hairpin RNA (shRNA) expression vector in an ESCC cell line.

EXPERIMENTAL DESIGN

Osteopontin mRNA expression was extracted from gene expression profiles of 23 tumors determined by cDNA microarray and was analyzed. Paraffin sections of 144 tumors were immunohistochemically investigated. Osteopontin protein expression in 34 cell lines was examined by Western blot. A doxycycline-inducible osteopontin-shRNA vector was stably transfected into HSA/c cells to assess the role of osteopontin in cell motility, invasion in vitro, tumor formation, and lymph node metastasis in nude mice.

RESULTS

cDNA microarray revealed that high osteopontin mRNA expression was associated with poor survival of ESCC patients (P = 0.029). In immunohistochemistry, osteopontin protein expression was associated with poor prognosis (P < 0.001), distant lymph node metastasis (P = 0.0004), tumor staging (P = 0.027), and histologic grade (P = 0.024). Multivariate analysis showed that osteopontin overexpression was the strongest independent prognostic factor among nine clinicopathologic variables (P < 0.001). Among cell lines tested, 30 had overexpressed osteopontin protein compared with a normal esophageal epithelial cell line. An inducible shRNA vector against osteopontin successfully down-regulated osteopontin expression by 71% to 88% and repressed cell motility by 69% to 97%, cell invasion by 59% to 71%, tumor formation by 56% to 92%, and lymph node metastasis by 50% to 67% in HSA/c cells.

CONCLUSIONS

Our findings suggest that osteopontin overexpression may play an important role in progression of ESCC and osteopontin could be a potential target of ESCC therapy.

摘要

目的

为阐明骨桥蛋白的临床意义以及食管鳞状细胞癌（ESCC）中骨桥蛋白条件性下调的作用，我们研究了肿瘤中骨桥蛋白的表达，并在一种ESCC细胞系中测试了一种可诱导的骨桥蛋白短发夹RNA（shRNA）表达载体。

实验设计

从通过cDNA微阵列确定的23个肿瘤的基因表达谱中提取骨桥蛋白mRNA表达并进行分析。对144个肿瘤的石蜡切片进行免疫组织化学研究。通过蛋白质印迹法检测34个细胞系中骨桥蛋白的蛋白表达。将一种强力霉素诱导的骨桥蛋白shRNA载体稳定转染至HSA/c细胞中，以评估骨桥蛋白在细胞运动、体外侵袭、肿瘤形成及裸鼠淋巴结转移中的作用。

结果

cDNA微阵列显示，骨桥蛋白mRNA高表达与ESCC患者的不良生存相关（P = 0.029）。在免疫组织化学中，骨桥蛋白蛋白表达与不良预后（P < 0.001）、远处淋巴结转移（P = 0.0004）、肿瘤分期（P = 0.027）及组织学分级（P = 0.024）相关。多变量分析显示，在9个临床病理变量中，骨桥蛋白过表达是最强的独立预后因素（P < 0.001）。在所测试的细胞系中，与正常食管上皮细胞系相比，30个细胞系的骨桥蛋白蛋白过表达。针对骨桥蛋白的可诱导shRNA载体成功将HSA/c细胞中的骨桥蛋白表达下调71%至88%，并将细胞运动抑制69%至97%，细胞侵袭抑制59%至71%，肿瘤形成抑制56%至92%，淋巴结转移抑制50%至67%。