下肢骨折患者miR-342-5P表达对其成骨细胞分化、增殖、迁移的影响及作用机制

Effects of mi R-342-5P expression on osteoblast differentiation,proliferation and migration in patients with lower extremity fractures and its mechanism

目的探讨下肢骨折患者miR-342-5P表达对其成骨细胞分化、增殖、迁移的影响及其作用机制。方法选取我院2019年1月至2020年12月下肢骨折患者35例,使用Percoll密度梯度离心法获取骨髓间充质干细胞样本并培养,诱导其成骨细胞分化,分别进行miR-342-5P转染、miR-342-5P沉默处理,并取无干预细胞作为对照(NC组),观察不同处理下样本细胞活力、分化、增殖、迁移相关因子表达;确认结果后进一步建立miR-342-5P转染+si-Bmp7组,观察不同处理下成骨细胞分化、增殖、迁移相关蛋白表达及MEK/ERK通路表达。结果过表达组细胞凋亡率、成骨细胞细胞活力显著高于NC组及沉默组(P<0.05),过表达miR-342-5p的细胞中PCNA、CyclinA、CyclinE1、CDK2、CyclinD1、CDK4的水平明显降低,miR-342-5p沉默细胞水平均升高(P<0.05);过表达miR-342-5p细胞相对迁移率、ALP蛋白、RUNX2 mRNA及Ocn m RNA、Bmp7 mRNA水平显着低于NC组(P<0.05),miR-342-5p沉默细胞相对迁移显着高于NC组(P<0.05);miR-342-5p沉默组ALP蛋白、RUNX2 mRNA及Ocn mRNA、Bmp7 mRNA水平显著高于NC组(P<0.05);miR-342-5p inhibitor+si-Bmp7组细胞相对迁移率、PCNA、CyclinA、CyclinE1、CDK2、CyclinD1、CDK4、RUNX2、Ocn蛋白水平低于miR-342-5p沉默组(P<0.05),细胞凋亡率显著高于miR-342-5p沉默组(P<0.05),miR-342-5p沉默后p-MEK和p-ERK的表达均升高(P<0.05),敲除Bmp-7后,升高被消除。结论miR-342-5p可通过下调Bmp7表达抑制下肢骨折患者成骨细胞增殖、迁移和分化;MEK/ERK通路参与了miR-342-5p的调控机制,miR-342-5p的独特作用可能为下肢骨折愈合提供新的治疗靶点。

Objective To explore the effect of miR-342-5P expression on osteoblast differentiation,proliferation and migration in patients with lower extremity fractures and its mechanism.Methods A total of 35 patients with lower extremity fractures in our hospital from January 2019 to December 2020 were selected.Bone marrow mesenchymal stem cell samples were obtained by Percoll density gradient centrifugation and cultured to induce osteoblast differentiation.Transfection and silencing of miR-342-5P were performed,and cells without intervention were taken as control(NC group)to observe the expression of cell viability,differentiation,proliferation,and migration-related factors under different treatments;miR-342-5P was further established after confirming the results.In the transfection+si-Bmp7 group,the expression of osteoblast differentiation,proliferation,migration-related proteins and MEK/ERK pathway expression under different treatments were observed.Results The apoptosis rate and osteoblast cell viability in the overexpression group were significantly higher than those in the NC group and the silence group(P<0.05).The levels of miR-342-5p silenced cells increased;the relative mobility,ALP protein,RUNX2 mRNA and Ocn m RNA,and Bmp7 mRNA levels of miR-342-5p overexpressed cells were significantly lower than those in NC group(P<0.05),and miR-342-5p silenced cells The relative migration was significantly higher than that in the NC group(P<0.05);The levels of ALP protein,RUNX2 mRNA and Ocn m RNA,and Bmp7 mRNA in the miR-342-5p silencing group were significantly higher than those in the NC group(P<0.05);The relative migration rate of cells in the miR-342-5p inhibitor+si-Bmp7 group,The protein levels of PCNA,CyclinA,CyclinE1,CDK2,CyclinD1,CDK4,RUNX2,and Ocn were lower than those in the miR-342-5p silence group(P<0.05),and the apoptosis rate was significantly higher than that in the miR-342-5p silence group(P<0.05).After miR-342-5p silencing,the expression of both-MEK and p-ERK was increased(P<0.05),and the increase was abolished after knockdown of Bmp-7.Conclusion The miR-342-5p can inhibit the proliferation,migration and differentiation of osteoblasts in patients with lower extremity fractures by down-regulating the expression of Bmp7.In addition,MEK/ERK pathway is involved in the regulatory mechanism of miR-342-5p,and the unique role of miR-342-5p It may provide a new therapeutic target for lower extremity fracture healing.