成骨不全XV型WNT1基因突变影响成骨细胞分化的机制

Osteogenesis imperfecta XV type WNT1 gene mutation affects the mechanism of osteoblast differentiation

目的探讨成骨不全(OI)XV型患者中,WNT1 c.110T>C/p.I37T和c.505G>T/p.G169C错义突变对WNT1/β-catenin信号通路影响成骨细胞增殖分化的机制。方法构建野生型WNT1、WNT1c.110T>C突变体、WNT1 c.505G>T突变体、WNT1 c.884C>A突变体(阳性对照)和空质粒(载体,阴性对照)转染成骨前细胞(MC3T3-E1)。MTS法检测WNT1基因突变对细胞活性的影响,实时定量PCR(qPCR)和免疫印迹法(WB)分别检测BMP2和RANKL(成骨细胞和破骨细胞分化标志物)的表达。采用qPCR、WB和免疫荧光(IF)检测成骨细胞中WNT1的m RNA、蛋白表达水平与WNT1/β-catenin信号通路相关的蛋白表达水平。结果转染24 h后,WNT1野生型细胞活性显著高于空载组、c.505G>T及c.884C>A突变组,转染48 h后,c.110T>C和c.505G>T突变细胞活性较WNT1野生型细胞显著降低,差异有统计学意义(P<0.05)。与野生型细胞株相比,c.110T>C和c.505G>T突变株中BMP2基因的表达显著水平降低,RANKL基因的表达显著升高,差异有统计学意义(P<0.05)。WNT1 mRNA在空载组,突变型组和野生型组中的表达量与空载组相比,WNT1在突变型组和野生型组中表达显著上升,差异有统计学意义(P<0.05)。与野生型细胞株相比,突变细胞株中非磷酸化β-catenin及磷酸化GSK-3β的表达水平较野生型显著下调,差异有统计学意义(P<0.05)。结论WNT1 c.110T>C和c.505G>T突变可能通过抑制WNT1/β-catenin信号通路影响成骨细胞增殖和成骨表型,这可能与OI有关。

Objective To explore the effects of WNT1 c.110 T>C/p.I37 T and c.505 G>T/p.G169 C missense mutations on the mechanism of the proliferation and differentiation of osteoblasts in the WNT1/β-catenin signaling pathway in XV patients with osteogenesis imperfecta(OI).Methods The wild-type WNT1,WNT1 c.110 T>C mutant,WNT1 c.505 G>T mutant,WNT1 c.884 C>A mutant(positive control),and empty vector(negative control)plasmids were constructed and transfected into preosteoblast(MC3 T3-E1)cells.MTS assay was used to assess the effect of WNT1 mutation on cell activity,and the expressions of BMP2 and RANKL(osteoblast and osteoclast differentiation markers)were detected using quantitative real-time PCR(RT-qPCR)and western blotting(WB).Finally,qPCR,WB,and immunofluorescence(IF)assays were used to detect m RNA,protein expression levels of WNT1,and proteins expression levels related to the WNT1/β-catenin signaling pathway in osteoblasts.Results Activity of Wnt1 wild-type cells was significantly higher than that of the empty vector group,c.505 G>T and c.884 C>a mutation groups at 24 h after transfection(P<0.05).The activity of c.110 T>C and c.505 G>T mutation cells were significantly lower than that of Wnt1 wildtype cells at 48 h after transfection(P<0.05).Compared with wild-type cell lines,the expression of BMP2 gene in c.110 T>C and c.505 G>T mutant strains significantly decreased,and the expression of RANKL gene significantly increased(P<0.05).The expression levels of Wnt1 m RNA in the empty vector group,mutant and wild type groups were,respectively.Compared with the empty vector group,the expression levels of Wnt1 m RNA in the mutant group and wild type group were significantly higher(P<0.05).Compared with the wild-type cell line,the expression levels of non-phosphorylatedβ-Catenin and phosphorylated GSK-3βin the mutant cell line were significantly lower than those in the wild-type cell line,the difference was statistically significant(P<0.05).Conclusion WNT1 c.110 T>C and c.505 G>T mutations may alter osteoblast proliferation and osteogenic phenotype related to OI byinhibiting the WNT1/β-catenin signaling pathway.