Abstract:

Objective: To study the effect of long non-coding RNA-EPS (lincRNA-EPS) on osteoclastogenesis of RAW264.7 cells induced by receptor activator for nuclear factor-κB ligand (RANKL) and lipopolysaccharide (LPS). Methods: RAW264.7 cell strain overexpressing lincRNA-EPS (lincRNA-EPS OE strand) and its negative control strain were generated by transfection of lincRNA-EPS overexpressing vector and negative control vector and subsequent screening. Osteogenesis was induced with either RANKL or LPS (after pretreatment with RANKL). The cells were then stained with tartrate resistant acid phosphatase staining (TRAP staining) kit to count the number and observe the morphology of osteoclasts. Phalloidin staining was performed to detect the F-actin rings and therefore to evaluate bone-resorbing ability of osteoclasts. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to measure the mRNA levels of osteoclast-associated genes. Results: RAW264.7 cell strain overexpressing lincRNA-EPS and its negative control strain were generated. Neither strain showed significant difference during osteoclastogenesis (P>0.05), either induced by RANKL or LPS; for osteoclastogenesis by LPS induction, TRAP staining showed that significantly more osteoclasts were generated from lincRNA-EPS OE strain (P<0.05), while phalloidin staining showed that larger F-actin rings were generated from lincRNA-EPS OE strain, and RT-qPCR also showed that the mRNA levels of 4 osteoclastogenesis-related genes (TRAP, CTSK, DC-STAMP and ATP6v0d2) after induction were significantly lower in lincRNA-EPS OE strain (P<0.05); however, those difference were not present (P>0.05) when osteoclastogenesis was induced by RANKL. Conclusion: The overexpression of lincRNA-EPS inhibits LPS-induced osteoclastogenesis and the function of osteoclasts. However, the overexpression of lincRNA-EPS does not affect RANKL-induced osteoclastogenesis significantly.

Key words: long non-coding RNA, periodontitis, osteoclastogenesis, lipopolysaccharide

摘要：

目的: 探究长链非编码RNA-EPS（long non-coding RNA-EPS，lincRNA-EPS）对核因子κB受体活化因子配体（receptor activator for nuclear factor-κB ligand，RANKL）和脂多糖（lipopolysaccharide，LPS）诱导RAW264.7细胞破骨分化的影响。方法: 构建过表达lincRNA-EPS的RAW264.7细胞株和阴性对照株，然后用RANKL诱导法和LPS诱导法（RANKL预刺激后行LPS诱导）同时进行破骨分化诱导，通过抗酒石酸酸性磷酸酶染色（tartrate resistant acid phosphatase staining，TRAP staining）观察产生破骨细胞数量和形态的差异，鬼笔环肽染色评估所产生破骨细胞功能的差异，实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）检测诱导后破骨相关基因mRNA表达量的差异。结果: 成功构建过表达lincRNA-EPS的RAW264.7细胞株和阴性对照株；不论是RANKL法诱导还是LPS法诱导，lincRNA-EPS过表达株和阴性对照株的细胞增殖情况均无显著差异（P>0.05）；LPS法诱导下，TRAP染色显示lincRNA-EPS过表达株分化出的破骨细胞数量显著少于阴性对照株（P<0.05），鬼笔环肽染色显示lincRNA-EPS过表达株产生的F-肌动蛋白环体积显著小于阴性对照株，RT-qPCR显示lincRNA-EPS过表达株的破骨相关基因TRAP、CTSK、DC-STAMP、ATP6v0d2 mRNA表达均显著低于阴性对照株（P<0.05）；但在RANKL法诱导下，二者各项表达均无显著差异（P>0.05）。结论: lincRNA-EPS的过表达对LPS诱导的破骨分化有明显的抑制作用，而且对诱导出的破骨细胞功能也起到负向调节作用，但lincRNA-EPS过表达对RANKL诱导的破骨分化及产生的破骨细胞的功能无显著影响。

关键词: 长链非编码RNA, 牙周炎, 破骨分化, 脂多糖

CLC Number: 

• R782