He Yu, Meng Chunqing, Shao Zengwu, Wang Hong, Yang Shuhua
Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P.R. China.
Cell Physiol Biochem. 2014;34(5):1485-96. doi: 10.1159/000366353. Epub 2014 Oct 8.
Osteosarcoma is the most common primary bone malignancy in children and adolescents, and the pathogenesis of this cancer remains unclear. Therefore, the discovery of new biomarkers for the diagnosis, prognosis, and treatment of osteosarcoma remains an important but unmet clinical need.
Quantitative real-time PCR was carried out to examine the expression of miR-23a. Methylation-specific PCR was performed to evaluate the DNA methylation status of the miR-23a promoter. Cell proliferation, migration, and invasion were examined by cell counting assays, wound healing assays, and cell invasion assays, respectively. Western blot analysis and luciferase reporter assays were performed to identify miR-23 target genes. Nude mice were used to investigate the function of miR-23a in vivo.
The expression of miR-23a was decreased in osteosarcoma cells and tissues compared to normal controls. The promoter region of the miR-23a gene was hypermethylated in osteosarcoma cells, and demethylase treatment increased the expression of miR-23a. The ectopic expression of miR-23a led to retarded proliferation, migration, and invasion of osteosarcoma cells, whereas the depletion of miR-23a resulted in the opposite effects. MiR-23a suppressed the transcription of RUNX2 and CXCL12 by binding to the 3' UTRs of these mRNAs. The cellular function of miR-23a is RUNX2/CXCL12-dependent, and the overexpression of RUNX2 or CXCL12 rescued the impaired cell growth, migration, and invasion induced by miR-23a. Nude mouse experiments indicated that miR-23a may inhibit the proliferation of osteosarcoma cells in vivo.
We identified miR-23a as a tumor suppressor in osteosarcoma. Our data clarify the mechanism of osteosarcoma progression and demonstrated the potential for exploiting miR-23a as a diagnostic marker for osteosarcoma.
骨肉瘤是儿童和青少年中最常见的原发性骨恶性肿瘤，其发病机制尚不清楚。因此，发现用于骨肉瘤诊断、预后和治疗的新生物标志物仍然是一项重要但尚未满足的临床需求。
采用定量实时聚合酶链反应检测miR-23a的表达。进行甲基化特异性聚合酶链反应以评估miR-23a启动子的DNA甲基化状态。分别通过细胞计数实验、伤口愈合实验和细胞侵袭实验检测细胞增殖、迁移和侵袭情况。进行蛋白质印迹分析和荧光素酶报告基因实验以鉴定miR-23的靶基因。使用裸鼠在体内研究miR-23a的功能。
与正常对照相比，骨肉瘤细胞和组织中miR-23a的表达降低。骨肉瘤细胞中miR-23a基因的启动子区域发生高甲基化，去甲基化酶处理可增加miR-23a的表达。miR-23a的异位表达导致骨肉瘤细胞增殖、迁移和侵袭受阻，而miR-23a的缺失则产生相反的效果。miR-23a通过与这些mRNA的3'非翻译区结合来抑制RUNX2和CXCL12的转录。miR-23a的细胞功能依赖于RUNX2/CXCL12，RUNX2或CXCL12的过表达挽救了miR-23a诱导的细胞生长、迁移和侵袭受损。裸鼠实验表明miR-23a可能在体内抑制骨肉瘤细胞的增殖。
我们确定miR-23a为骨肉瘤中的一种肿瘤抑制因子。我们的数据阐明了骨肉瘤进展的机制，并证明了将miR-23a用作骨肉瘤诊断标志物的潜力。
