长链非编码RNA SATB2-AS1靶向SATB2调控骨肉瘤发生发展的机制研究

Osteosarcoma has a poor clinical outcome, and therefore, early diagnosis and effective therapy becomes critical. Long non-coding RNAs (lncRNAs) are important regulator of gene expression and closely related to tumor development. Using lncRNA-CHIP and qPCR, we have demonstrated that level of lncRNA SATB2-AS1 (special AT-rich sequence binding protein 2 - antisense RNA 1) is abnormally high in osteosarcoma. Functional studies reveled that SATB2-AS1 promoted osteosarcoma development and migration by increasing the expression of SATB2 protein. A detailed bioinformatic analysis showed SATB2-AS1 to be a natural antisense transcript of SATB2 mRNA and therefore might regulate the expression of SATB2. Moreover, SATB2 is known to promote osteoblast differentiation, bone formation, and osteosarcoma invasion via EPLIN (Epithelial Protein Lost In Neoplasm) and cytoskeleton. Keeping this background in mind, we propose that SATB2-AS1 can form double-stranded RNA with SATB2 mRNA and therefore enhancing the expression of SATB2 that might contribute to osteosarcoma development via EPLIN and cytoskeleton. To test this hypothesis, fistly we will analyze the correlations of the expression levels of SATB2-AS1 and SATB2 with the clinicopathologic parameters of osteosarcoma, such as patient age, patient sex, tumor size, stage, metastasis and recurrence of the disease. Then we will manipulate the expression of SATB2-AS1 in osteosarcoma cell lines and will determine the changes in the cell growth, invasion, migration, as well as the expression of EPLIN and cytoskeleton structure. If we prove our hypothesis in vitro, we would like to test this hypothesis in vivo using mouse model. We will use nude mice bearing osteosarcoma as in vivo models to observe the relationships among SATB2-AS1, SATB2 and tumor biological behavior. In order to determine the exact site(s) and mechanism how SATB2-AS1 promotes the expression of SATB2, we will perform RNA pull-down, RNase protection assay and segmented RNA interference. In summary, the project will find out the regulatory role of SATB2-AS1 on osteosarcoma development, making SATB2-AS1 a promising clinical diagnostic biomarkers. Meanwhile, in-depth study will lead to the regulatory mechanism of SATB2-AS1 and provide a new target for the treatment of osteosarcoma.

骨肉瘤易复发，转移率高，疗效差，开发新诊治方法是临床急需。前期研究发现长链非编码RNA SATB2-AS1在骨肉瘤中高表达，可促进骨肉瘤细胞生长、迁移，并促进SATB2蛋白表达。SATB2-AS1是SATB2的天然反义转录物，SATB2促进成骨分化和骨形成，经由EPLIN和细胞骨架调控骨肉瘤。由此本课题组提出，SATB2-AS1通过促进SATB2的表达，经EPLIN和细胞骨架促进骨肉瘤的发生发展。本项目拟检测骨肉瘤标本中SATB2-AS1和 SATB2的表达，结合临床数据分析其与肿瘤分期、预后的关系；构建SATB2-AS1高、低表达骨肉瘤细胞株，检测细胞生长、侵袭及EPLIN和细胞骨架变化；进一步改变SATB2的表达，观察能否逆转其生物效应，并在裸鼠体内验证；最后应用RNA pull-down等实验明确SATB2-AS1促进SATB2表达的作用机制，有望为骨肉瘤早期诊断和治疗提供新靶点。

本研究前期研究发现长链非编码RNA SATB2-AS1在骨肉瘤中高表达，可促进骨肉瘤细胞生长、迁移。本研究首先扩大临床样品量，实时定量RT-PCR检测骨肉瘤组织及瘤旁组织中SATB2-AS1的表达量，并与临床病理分期、转移、预后等关联分析。发现与瘤旁组织比较，SATB2-AS1在骨肉瘤组织中高表达，其表达量与是否有远处转移呈正相关。进一步我们利用慢病毒感染构建SATB2-AS1敲低和高表达稳转骨肉瘤细胞株，细胞功能检测表明，SATB2-AS1过表达能促进骨肉瘤HOS和U2OS细胞的生长和修复，而敲低SATB2-AS1表达则抑制HOS和U2OS的生长和修复。裸鼠体内成瘤实验表明，SATB2-AS1 SiRNA干扰后的hos细胞不能成瘤或成瘤重量明显低于非干扰组和SATB2-AS1高表达組；SiRNA干扰SATB2-AS1的表达，U2OS和HOS两组细胞均呈现细胞早期凋亡都增加，反之SATB2-AS1可以抑制骨肉瘤细胞的凋亡。细胞周期研究结果显示SATB2-AS1 siRNA干扰后的细胞均呈现G0/G1期升高，S期明显减少，说明SATB2-AS1沉默后细胞呈现G0/G1阻滞，细胞复制受到抑制。进一步机制研究结果表明，SATB2-AS1在细胞质和细胞核中均有分布，主要分布于细胞质，约占总量的80%；无论是在HOS细胞还是U2OS细胞中，SATB2-AS1的降低或升高，SATB2 mRNA的表达基本保持稳定，而在SATB2-AS1高表达的HOS和U2OS细胞中，SATB2蛋白表达量明显升高，SATB2-AS1 低表达则SATB2蛋白表达量明显减少；双荧光素酶报告基因试验验证SATB2的启动子区域与SATB2-AS1 有结合。以上结果说明，SATB2-AS1通过调控基因SATB2的翻译进而调控骨肉瘤细胞的生长与转移。本项目发现了SATB2-AS1在骨肉瘤中高表达，并对通过调控基因SATB2的翻译调控骨肉瘤生长、转移。有望为骨肉瘤早期诊断和治疗提供新靶点。