LncRNA ZBED3-AS1/miR-339-5p/Notch 1轴调控骨质疏松大鼠成骨细胞增殖分化

目的探究LncRNA ZBED3-AS1调控miR-339-5p/Notch 1在骨质疏松大鼠成骨细胞增殖分化中的作用。方法构建假手术(Sham)组和模型(Model)组大鼠模型,对大鼠的骨密度进行检查观测大鼠建模情况。分离大鼠成骨细胞,qRT-PCR检测细胞中ZBED3-AS1、miR-339-5p的表达。对Sham组和Model组大鼠成骨细胞进行分组后转染。CCK8、茜素红(AR-S)染色、碱性磷酸酶(ALP)染色检测各组细胞增殖分化能力。FISH实验测定ZBED3-AS1在细胞中的分布。双荧光素酶报告证实ZBED3-AS1与miR-339-5p及miR-339-5p和Notch 1之间的关系。Western blot检测Notch通路相关因子的表达。结果检测股骨骨密度发现,Model组大鼠骨密度明显低于Sham组大鼠(P=0.0 057)。与Sham组大鼠比较,Model组大鼠成骨细胞中ZBED3-AS1呈低表达,而miR-339-5p呈高表达(均P<0.05)。过表达ZBED3-AS1能促进成骨细胞的增殖分化;而敲减ZBED3-AS1则能抑制成骨细胞增殖分化(均P<0.05)。ZBED3-AS1作为ceRNA调控miR-339-5p(均P<0.05)。过表达miR-339-5p能抑制成骨细胞的增殖分化能力,而该作用可被ZBED3-AS1过表达部分挽救。Notch 1被证实为miR-339-5p的作用靶点,且干预ZBED3-AS1/miR-339-5p的表达能影响Notch 1的蛋白表达,ZBED3-AS1/miR-339-5p对成骨细胞的调控可能是通过Notch通路实现的。结论 ZBED3-AS1能作为ceRNA调控miR-339-5p,进而影响骨质疏松大鼠成骨细胞增殖分化。

LncRNA ZBED3-AS1/miR-339-5p/Notch 1 axis regulates osteoblast proliferation and differentiation in osteoporotic rats

Objective To investigate the role of LncRNA ZBED3-AS1 in osteoblast proliferation and differentiation in osteoporotic rats through regulating miR-339-5p/Notch 1.Methods The rat models of sham operation(Sham)group and model(Model)group were established,and the bone mineral density(BMD)of rats was examined.Rat osteoblasts were isolated and the expression of ZBED3-AS1 and miR-339-5p was detected by qRTPCR.The osteoblasts of rats in Sham group and Model group were divided into different groups and transfected.CCK8,alizarin red(AR-S)staining and alkaline phosphatase(ALP)staining were used to detect the proliferation and differentiation ability of cells in each group.The distribution of ZBED3-AS1 in cells was determined by FISH assay.Double luciferase report confirmed the relationship between ZBED3-AS1 and miR-339-5p as well as miR-339-5p and Notch 1.Western blot was used to detect the expression of Notch pathway related factors.Results The bone mineral density of femur in Model group was significantly lower than that in Sham group(P=0.0057).Compared with Sham group,the expression of ZBED3-AS1 in osteoblasts of Model group was lower,while that of miR-339-5p was higher(all P<0.05).Overexpression of ZBED3-AS1 could promote the proliferation and differentiation of osteoblasts,while knockdown of ZBED3-AS1 could inhibit the proliferation and differentiation of osteoblasts(all Pv0.05).ZBED3-AS1 could regulate miR-339-5p as ceRNA(all P<0.05).Overexpression of miR-339-5p can inhibit the proliferation and differentiation of osteoblasts,which can be partially saved by overexpression of ZBED3-AS1.Notch 1 was confirmed as a target of miR-339-5p,at the same time,interfering with the expression of ZBED3-AS1/miR-339-5p can affect the expression of Notch-1 protein,and the regulation of ZBED3-ASl/miR-339-5p on osteoblasts may be realized through Notch pathway.Conclusion ZBED3-AS1 can be used as ceRNA to regulate miR-339-5p,and then affect the proliferation and differentiation of osteoblasts in osteoporotic rats.