Supplementary Material for: Rat Osteosarcoma Cells as a Therapeutic Target Model for Osteoregeneration via Sclerostin Knockdown

There are various conceptually different strategies to improve bone regeneration and to treat osteoporosis, each with distinct inherent advantages and disadvantages. The use of RNA interference strategies to suppress the biological action of catabolic factors or antagonists of osteogenic proteins is promising, and such strategies can be applied locally. They are comparably inexpensive and do not suffer from stability problems as protein-based approaches. In this study, we focus on sclerostin, encoded by the SOST gene, a key regulator of bone formation and remodeling. Sclerostin is expressed by mature osteocytes but also by late osteogenically differentiated cells. Thus, it is difficult and requires long-term cultures to investigate the effects of SOST silencing on the expression of osteogenic markers using primary cells. We, therefore, selected a rat osteosarcoma cell line, UMR-106, that has been shown to express SOST and secrete sclerostin in a comparable fashion as late osteoblasts and osteocytes. We investigated the effects of differentiating supplements on SOST expression and sclerostin secretion in UMR-106 cells and found that addition of 100 ng/ml of bone morphogenetic protein (BMP)-2 strongly induced sclerostin secretion, whereas dexamethasone inhibited secretion. Effects of silencing SOST in UMR-106 cells cultured in various differentiation media including BMP-2 and/or dexamethasone were determined next with the aim to find promising test conditions for a readout system for the evaluation of future small interfering RNA release formulations for local induction of bone formation. We found a direct correlation between attenuated SOST expression and an increase in the osteogenic potential of UMR-106 cells. The combination of SOST silencing and BMP-2 could synergistically improve osteogenic factors. A lowered proliferation rate in silenced groups may indicate a faster switch to differentiation.

当前用于促进骨再生与治疗骨质疏松症的策略在概念上存在诸多差异，各类策略均有其独特的固有优缺点。采用RNA干扰（RNA interference）策略抑制分解代谢因子或成骨蛋白拮抗剂的生物学活性，极具应用前景，且该类策略可实现局部给药；其成本相对低廉，同时不会像蛋白类疗法那样面临稳定性难题。本研究聚焦于骨硬化蛋白（sclerostin）——其由SOST基因编码，是骨形成与骨重塑的关键调控因子。骨硬化蛋白不仅由成熟骨细胞表达，也可在成骨分化晚期细胞中产生。因此，若采用原代细胞开展SOST沉默对成骨标志物表达影响的相关研究，不仅操作难度较高，还需要进行长期的体外培养。为此我们选取了大鼠骨肉瘤细胞系UMR-106，该细胞系已被证实能够表达SOST基因，并以与晚期成骨细胞及骨细胞相似的模式分泌骨硬化蛋白。我们研究了分化添加剂对UMR-106细胞中SOST表达及骨硬化蛋白分泌的影响，发现添加100 ng/ml的骨形态发生蛋白（bone morphogenetic protein, BMP）-2可显著诱导骨硬化蛋白分泌，而地塞米松（dexamethasone）则会抑制该分泌过程。接下来，我们测定了在包含BMP-2和/或地塞米松的多种分化培养基中培养的UMR-106细胞中，SOST沉默的相关效应，旨在为后续用于评估局部诱导骨形成的小干扰RNA（small interfering RNA, siRNA）递送制剂的检测系统，筛选出合适的测试条件。我们发现，SOST表达的减弱与UMR-106细胞成骨潜能的提升存在直接相关性。SOST沉默与BMP-2的联合应用可协同提升成骨相关因子的表达水平。沉默组细胞的增殖速率降低，这或许表明其向分化状态的转换更快。