MiR-367通过Wnt/β-catenin通路抑制成骨细胞增殖的研究

MiR-367 Inhibits Osteoblast Proliferation through Wnt/β-catenin Pathway

目的探究MiR-367通过Wnt/β-连环蛋白(β-catenin)通路对成骨细胞增殖的抑制作用。方法培养骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)后转染miR-367模拟物或抑制物,茜素红染色观察成骨分化;培养新生大鼠成骨细胞,转染miR-367模拟物或抑制物、联合使用β-catenin激动剂SKL2001或抑制剂XAV939,采用MTT法检测细胞增殖活力,采用荧光定量PCR及Western blot检测β-catenin及成骨标志基因Runt2相关转录因子2(Runt-related transcription factor 2,RUNX2)、碱性磷酸酶(alkaline phosphatase,ALP)、骨钙素(osteocalcin,OCN)、Ⅰ型胶原(Collagen-Ⅰ,Col-Ⅰ)的表达。结果转染miR-367模拟物或抑制物后,BMSCs茜素红染色的OD570值与转染NC模拟物或抑制物比较差异无统计学意义(P>0.05);转染miR-367模拟物后新生大鼠成骨细胞MTS检测的OD490值及β-catenin、RUNX2、ALP、OCN、Col-Ⅰ的表达水平明显低于转染NC模拟物,转染miR-367抑制物后新生大鼠成骨细胞MTS检测的OD490值及β-catenin、RUNX2、ALP、OCN、Col-Ⅰ的表达水平明显高于转染NC抑制物(P<0.05);转染miR-367模拟物联合SKL2001处理后新生大鼠成骨细胞中β-catenin、RUNX2、ALP、OCN、Col-Ⅰ的表达水平明显高于转染miR-367模拟物,转染miR-367抑制物联合XAV939处理后新生大鼠成骨细胞中β-catenin、RUNX2、ALP、OCN、Col-Ⅰ的表达水平明显低于转染miR-367抑制物。结论 miR-367能够抑制成骨细胞的增殖、但不影响成骨细胞的分化,这一作用可能与抑制Wnt/β-catenin通路有关。

Objective To study the inhibitory effect of MiR-367 on osteoblast proliferation through Wnt/β-catenin pathway.Methods Bone marrow mesenchymal stem cells(BMSCs)were transfected with mimic or inhibitor of miR-367,and osteogenic differentiation was observed by alizarin red staining.Osteoblasts from neonatal rats were cultured and transfected with mimic or inhibitor of miR-367,combined with SKL2001 or XAV939 as an agonist or antagonist ofβ-catenin.Cell proliferation activity was measured by MTT method,and the expression ofβ-catenin and osteogenic markers gene RUNX2,ALP,OCN,Col-Ⅰwere detected by fluorescence quantitative PCR and Western blot.Results After transfection of mir-367 mimics or inhibitor,OD570 value of alizarin red staining had no significant difference with those transfected with NC mimic or inhibitor(P>0.05).OD490 value of MTS and expression levels ofβ-catenin,RUNX2,ALP,OCN and Col-Ⅰin osteoblasts of neonatal rats after transfection of miR-367 mimic were significantly lower than those of NC mimic,OD490 value of MTS and expression levels ofβ-catenin,RUNX2,ALP,OCN and Col-Ⅰin osteoblasts of neonatal rats after transfection of mir-367 inhibitor were significantly higher than those of NC inhibitor(P<0.05).The expression levels ofβ-catenin,RUNX2,ALP,OCN and Col-Ⅰin osteoblasts of newborn rats treated with miR-367 mimic combined with SKL2001 were significantly higher than those of miR-367 mimic.The expression levels ofβ-catenin,RUNX2,ALP,OCN and Col-Ⅰin osteoblasts of newborn rats treated with miR-367 inhibitor combined with SKL2001 were significantly lower than those of miR-367 inhibitor.Conclusion MiR-367 can inhibit the proliferation of osteoblasts,but does not affect the differentiation of osteoblasts,which may be achieved by inhibiting Wnt/β-catenin pathway.