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

doi:10.3969/j.issn.2095-4344.1429

Abstract

Abstract:

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.

Key words: osteosarcoma, periostin, POSTN protein, angiogenesis, KDR, PI3K/AKT pathway, Saos-2 cells, platelet endothelial cell adhesion molecule 1

CLC Number:

Xu Chaojian, Zhang Long, Cheng Caitong, Feng Yi, Lü Jia, Sun Xiaojuan, Lü Zhi .

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

[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(35): 5638-5644.

Figures/Tables 3

2.1 体外实验结果 2.1.1 临床骨肉瘤样本中POSTN和KDR表达 qRT-PCR检测结果显示，人骨肉瘤组织中的POSTN和KDR mRNA表达水平均明显高于正常骨组织(P < 0.01)，见图1A。Western blot检测同样显示POSTN和KDR蛋白在人骨肉瘤组织中的表达水平高于正常骨组织，见图1B。 2.1.2 人骨肉瘤细胞系中的POSTN mRNA表达相对于c-28细胞，骨肉瘤细胞系中的POSTN mRNA表达升高(P < 0.01)，且在Saos-2细胞中的POSTN mRNA表达高于MG-63和U2-OS细胞，见图2。后续以Saos-2细胞为实验对象。 2.1.3 转染效率检测转染48 h后倒置荧光显微镜下观测荧光细胞数量，结果显示两组转染效率均在80%以上，见图3A。各组细胞传代一二次后部分细胞冻存于液氮中，另有部分细胞用于RNA提取，qRT-PCR检测POSTN的mRNA表达水平，结果显示POSTN靶点2序列(target 2)质粒的沉默效率最高(P < 0.05)，见图3B。 2.2 体内实验结果 2.2.1 成瘤情况 Saos-2细胞混和Matrigel混悬液原位注射于裸鼠胫骨髓腔内，小动物X射线扫描针头位置，确保细胞混悬液注射入胫骨髓腔内，12只裸鼠全部成瘤，见图4。 2.2.2 移植瘤体积与质量根据移植瘤裸鼠的瘤体长宽度测量数据绘制生长曲线，接种5周后完整取出瘤体，包括胫骨部分但需切除瘤体未包裹的胫骨及裸鼠脚，结果显示实验组瘤体体积和质量均小于对照组，见图5。 2.2.3 血管生成接种3周后尾静脉注射AngioSense 750 EX荧光检测液，注射48 h后进行小动物活体成像扫描，显示实验组瘤体内血管生成量较对照组明显减弱(P < 0.05)，见图6。 2.2.4 蛋白检测实验组中POSTN、增殖细胞核抗原、KDR、p-AKT蛋白表达量均较对照组减弱(P < 0.05)，见图7，提示沉默POSTN可显著抑制PI3K/AKT的磷酸化过程。 2.2.5 病理观察裸鼠瘤体组织石蜡切片苏木精-伊红染色可见致密的粉红色、多型性类骨质；免疫组织化学染色显示，实验组中内皮细胞黏附分子表达较对照组减少，见图8。"

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