shRNA沉默骨膜蛋白基因抑制人骨肉瘤的血管生成

Silencing of periosteal protein gene by shRNA inhibits angiogenesis in human osteosarcoma

背景:骨膜蛋白(periostin,POSTN)是一种细胞外基质蛋白,在多种肿瘤组织中表达上调,与肿瘤的迁移、增殖和血管生成能力有关。血管内皮生长因子受体2(vascularendothelialgrowthfactor2,VEGFR2/KDR)与其配体血管内皮生长因子结合后主要调控血管的发生和构建,但KDR与POSTN在骨肉瘤中的相互作用尚不清楚。目的:探讨POSTN在体内、外水平对人骨肉瘤增殖和体内血管生成能力影响及其作用机制。方法:①体外实验:qRT-PCR和Westernblot检测人骨肉瘤临床标本组织中POSTN和KDR的mRNA及蛋白表达水平;qRT-PCR检测3株不同人骨肉瘤细胞系内POSTN表达,筛选基因表达最高的细胞系进行转染实验;转染慢病毒包装pPLK-POSTN-shRNA进入Saos-2细胞,选出3个作用靶点中抑制率最高的序列进行后续实验;②体内实验:取裸鼠(购自北京维通利华实验动物技术有限公司)12只,实验组(n=6)将慢病毒包装pPLK-POSTN-shRNA转染的Saos-2细胞注射至胫骨髓腔中,对照组(n=6)将慢病毒包装pPLK-Scramble-shRNA转染的Saos-2细胞注射至胫骨髓腔中,接种5周后,测量瘤体体积与质量;接种3周后,应用小动物活体成像检测血管生成情况;接种5周后取瘤体组织,Westernblot检测POSTN、增殖细胞核抗原、KDR和p-AKT蛋白表达,病理切片免疫组织化学染色内皮细胞黏附分子蛋白表达。实验方案经山西医科大学第二医院伦理委员会批准(编号:2018002)。结果与结论:①体外实验:骨肉瘤组织中POSTN和KDR的mRNA及蛋白水平表达均高于正常骨组织(P<0.01);Saos-2细胞中的POSTN基因表达量较MG-63和U2-OS细胞高;②体内实验:实验组瘤体体积和质量均小于对照组(P<0.05);实验组瘤体内血管生成量少于对照组(P<0.05);实验组POSTN、增殖细胞核抗原、KDR和p-AKT及内皮细胞黏附分子蛋白表达均低于对照组(P<0.05);③结果表明:沉默POSTN基因可抑制骨肉瘤血管生成,其机制可能与KDR/PI3K/AKT通路活化有关。

BACKGROUND: Periostin(POSTN) is an extracellular matrix protein that is upregulated in most tumor tissues and is associated with tumor migration, proliferation and angiogenesis. Vascular endothelial growth factor receptor 2(VEGFR2/KDR) mainly regulates the occurrence and construction of blood vessels after combining with its ligand VEGF, but the interaction between KDR and POSTN in osteosarcoma is still unclear. OBJECTIVE: To investigate the effect of POSTN on the proliferation and angiogenesis of human osteosarcoma in vitro and in vivo and the underlying action mechanism. METHODS: In vitro study: The mRNA and protein levels of POSTN and KDR in human osteosarcoma clinical specimens were detected by qRT-PCR and western blot analysis. POSTN expression in three human osteosarcoma cell lines was detected by qRT-PCR. POSTN shRNA plasmids were transfected into Saos-2 cells and the sequence with the highest inhibition rate among the three targets was selected for subsequent experiments. Fluorescence quantity of cells was observed under fluorescence microscope, and the transfection efficiency was detected by qRT-PCR. In vivo study: twelve nude mice(Beijing Vital River Laboratory Animal Technology Co., Ltd., China) were randomly divided into an experimental group and a control group(n = 6/group). In the experimental group, Saos-2 cells transfected with pPLK-POSTN-shRNA were injected into the tibial medullary cavity of nude mice. In the control group, Saos-2 cells transfected with pPLK-Scramble-shRNA were identically injected. After 3 weeks of inoculation, angiogenesis was detected by in vivo imaging. After 5 weeks of inoculation, the removed tumor tissue was measured in volume and mass and then used for detecting the expression of POSTN, proliferating cell nuclear antigen, KDR and p-AKT protein by western blot analysis and the expression of endothelial cell adhesion molecule by immunohistochemical staining. This study was approved by Medical Ethics Committee, the Second Hospital of Shanxi Medical University of China(approval No. 2018002). RESULTS AND CONCLUSION:(1) In vitro study: The mRNA and protein levels of POSTN and KDR in osteosarcoma tissues were higher than those in normal bone tissues(P < 0.01). POSTN expression in Saos-2 cells was higher than that in MG-63 and U2-OS cells.(2) In vivo study: the tumor volume and mass of the experimental group were lower than those of the control group(P < 0.05). Osteosarcoma angiogenesis in the experimental group was significantly lower than that in the control group(P < 0.05). POSTN, proliferating cell nuclear antigen, KDR and p-Akt as well as endothelial cell adhesion molecule protein levels in the experimental group were significantly lower than those in the control group(P < 0.05).(3) These results suggest that silencing POSTN gene can inhibit osteosarcoma angiogenesis, which may be related to the activation of KDR/PI3K/AKT pathway.