Gong Yubao, Wei Zhengren, Liu Jianguo
Department of Orthopedics, The First Hospital of Jilin University, Changchun 130021, People's Republic of China.
Department of Pharmacology, Basic Medical School, Jilin University, Changchun 130021, People's Republic of China.
Onco Targets Ther. 2021 Jan 6;14:15-27. doi: 10.2147/OTT.S282485. eCollection 2021.
Osteosarcoma is the most common bone tumor and is characterized by the presence of malignant mesenchymal cells produced in the bone stroma. MiRNAs are known to function as post-transcriptional negative regulators of gene expression. Emerging evidence showed that miR-1225-5P functions as a tumor suppressor in several types of cancers. The detailed mechanisms of which miR-1225-5P suppresses tumor growth are not fully understood. The objective of the present study was to test the hypothesis that miR-1225-5P inhibits osteosarcoma cell growth in vitro and tumor growth in vivo by targeting YWHAZ expression.
Real-time PCR and Western blot were carried out to test the expression of miR-1225-5P and YWHAZ in osteosarcoma cell lines. Luciferase assay was used to demonstrate whether miR-1225-5P targets YWHAZ 3' UTR. To assess the function of miR-1225-5P in human osteosarcoma cell lines, gain-of-function and loss-of-function of miR-1225-5P were performed by transfecting miR-1225-5P mimic or miR-1225-5P inhibitor into osteosarcoma cell lines. Furthermore, cell cycle analysis was performed to elucidate the possible mechanisms of the action of miR-1225-5P and YWHAZ in human osteosarcoma cells. The potential therapeutic effect of miR-1225-5p was tested in human osteosarcoma xenograft mouse model, by intravenous injection of miR-1225-5P into nude mice. Tumor sizes were measured and lung metastasis was counted after the mice were sacrificed.
The expression of miR-1225-5P was inversely correlated with the expression of YWHAZ in human osteosarcoma cell lines. Database search revealed that miR-1225-5P targeted YWHAZ 3' UTR. Transfection of miR-1225-5P mimic downregulated YWHAZ expression, which was demonstrated by real-time PCR, Western blot and luciferase assay. Over-expression of miR-1225-5P reduced human osteosarcoma cell growth, migration and invasion by downregulating YWHAZ expression. Cell growth, migration and invasion were increased by inhibiting miR-1225-5P in human osteosarcoma cells. The inhibition of cell growth, migration and invasion was rescued by over-expression of YWHAZ in osteosarcoma cells. Cell cycle analysis revealed that miR-1225-5P inhibited G1/G0 phase exit. In vivo xenograft model demonstrated that miR-1225-5P inhibited in vivo osteosarcoma tumor growth and lung metastasis.
Our findings suggested that miR-1225-5P inhibits osteosarcoma cell growth in vitro and tumor growth in vivo by targeting YWHAZ. This study suggested that miR-1225-5P can serve as a potential therapeutic method for treating osteosarcoma.
骨肉瘤是最常见的骨肿瘤，其特征是在骨基质中产生恶性间充质细胞。已知微小RNA（miRNA）作为基因表达的转录后负调控因子发挥作用。新出现的证据表明，miR-1225-5P在几种类型的癌症中起肿瘤抑制作用。miR-1225-5P抑制肿瘤生长的详细机制尚未完全了解。本研究的目的是验证miR-1225-5P通过靶向14-3-3ζ（YWHAZ）表达来抑制骨肉瘤细胞体外生长和体内肿瘤生长这一假设。
采用实时定量聚合酶链反应（Real-time PCR）和蛋白质免疫印迹法检测骨肉瘤细胞系中miR-1225-5P和YWHAZ的表达。荧光素酶报告基因检测用于证明miR-1225-5P是否靶向YWHAZ的3'非翻译区（UTR）。为评估miR-1225-5P在人骨肉瘤细胞系中的功能，通过将miR-1225-5P模拟物或miR-1225-5P抑制剂转染到骨肉瘤细胞系中，分别进行miR-1225-5P的功能获得和功能丧失实验。此外，进行细胞周期分析以阐明miR-1225-5P和YWHAZ在人骨肉瘤细胞中的可能作用机制。通过向裸鼠静脉注射miR-1225-5P，在人骨肉瘤异种移植小鼠模型中测试miR-1225-5p的潜在治疗效果。处死小鼠后测量肿瘤大小并计数肺转移情况。
在人骨肉瘤细胞系中，miR-1225-5P表达与YWHAZ表达呈负相关。数据库搜索显示miR-1225-5P靶向YWHAZ的3'UTR。miR-1225-5P模拟物转染下调了YWHAZ表达，实时定量聚合酶链反应、蛋白质免疫印迹法和荧光素酶报告基因检测均证实了这一点。miR-1225-5P的过表达通过下调YWHAZ表达降低了人骨肉瘤细胞的生长、迁移和侵袭能力。在人骨肉瘤细胞中抑制miR-1225-5P可增加细胞生长、迁移和侵袭能力。在骨肉瘤细胞中过表达YWHAZ可挽救对细胞生长、迁移和侵袭的抑制作用。细胞周期分析显示miR-1225-5P抑制G1/G0期退出。体内异种移植模型表明miR-1225-5P抑制体内骨肉瘤肿瘤生长和肺转移。
我们的研究结果表明，miR-1225-5P通过靶向YWHAZ抑制骨肉瘤细胞体外生长和体内肿瘤生长。本研究表明，miR-
