糖基化终末产物刺激下长链非编码RNA H19通过WNT/β- catenin信号通路调控MC3T3细胞骨向分化

Long non-coding RNA H19 regulates the osteogenic differentiation of MC3T3-E1 cells through WNT/β-catenin signaling pathway under the stimulation of AGEs

目的:探究长链非编码RNA H19(LncRNA H19)在糖基化终末产物(AGEs)刺激下对小鼠前成骨细胞MC3T3-E1成骨分化的调控作用及其可能的机制。方法:10μg/mL AGEs浓度刺激下培养MC3T3-E1细胞,7 d后碱性磷酸酶染色、21 d后茜素红染色观察细胞矿化情况;RT-PCR检测细胞中ALP、Runx-2、OCN、SP7以及LncRNA H19 mRNA的表达水平,Western blotting检测细胞中Runx-2蛋白表达水平;采用细胞转染技术过表达LncRNA H19,RT-PCR检测LncRNA H19的转染效率;并检测转染LncRNA H19后细胞中骨基因的表达水平、ALP染色情况以及β-catenin的表达情况。结果:AGEs刺激下MC3T3-E1细胞碱性磷酸酶染色变浅、钙化结节形成减少,ALP、Runx-2、OCN、SP7、LncRNA H19基因表达水平降低(P<0.05),Runx-2蛋白水平表达下降;转染过表达LncRNA H19(lv-H19)后,LncRNA H19表达水平显著上调(P<0.05),ALP、Runx-2、OCN、SP7基因表达水平上升(P<0.05),碱性磷酸酶染色变深,β-catenin的表达增加。结论:AGEs刺激下,LncRNA H19可能通过WNT/β-catenin的表达影响了MC3T3-E1细胞的骨向分化。

Objective:To explore the regulatory effects of long non-coding RNA H19(LncRNA H19)on the osteogenic differentiation of MC3T3-E1 cells under the stimulation of advanced glycation end products(AGEs).Methods:MC3T3-E1 cells were cultured under the stimulation of 10μg/mL AGEs.The cells were observed by alkaline phosphatase(ALP)staining after 7 d and alizarin red staining after 21 d culture respectively.Cell transfection technology was used to overexpress LncRNA H19 in the cells,RT-qPCR was used to detect the mRNA expression levels of ALP,Runx-2,OCN,SP7 before and after transfection of LncRNA H19.Western blotting was used to detect the protein expression of Runx-2 andβ-catenin in the cells.Results:Under the stimulation of AGEs,the ALP staining color of MC3T3-E1 cells became lighter,the formation of calcified nodules was reduced,the mRNA expression levels of ALP,Runx-2,OCN,SP7 and LncRNA H19 were decreased(P<0.05),and the protein expression level of Runx-2 was decreased.After transfection of LncRNA H19(lv-H19),the mRNA expression of LncRNA H19 was significantly up-regulated(P<0.05),the mRNA expression of ALP,Runx-2,OCN and SP7 genes was increased(P<0.05),ALP staining was deeper,and the expression ofβ-catenin was increased.Conclusion:Under the stimulation of AGEs,LncRNA H19 may affect the osteogenic differentiation of MC3T3-E1 cells by activating the expression of WNT/β-catenin.