miR-16-5p调控Wnt/β-catenin通路活性在力学载荷促进骨形成中作用的小鼠在体研究

In vivo study on the role of miR-16-5p regulate Wnt/β-catenin pathway activity in bone formation induced by mechanical loading in mice

目的 探究LRP6是否为miR-16-5p靶基因及miR-16-5p调控Wnt/β-catenin通路活性在力学载荷促进小鼠骨形成中的作用。方法 利用microRNA信息库预测miR-16-5p的靶基因,行荧光素酶报告实验来验证LRP6是否为miR-16-5p的靶基因。构建跑台组和对照组小鼠验证不同力学环境下骨组织中miR-16-5p及LRP6是否差异表达。制造miR-16-5p差异表达及力学加载动物模型:正常+miRNA Agomir control组(A组)、正常+miR-16-5p Agomir组(B组)、跑台+miRNA Agomir Control组(C组)及跑台+miR-16-5p Agomir组(D组)。干预完成后处死小鼠获取骨组织,检测各组LRP6、β-catenin及Runx2 mRNA及蛋白表达变化,行股骨远端Micro-CT扫描及股骨干生物力学测试检测骨量及力学性能变化。结果 microRNA信息库显示LRP6为miR-16-5p的可能靶基因,荧光素酶报告实验验证LRP6是miR-16-5p的靶基因。与对照组相比,跑台组小鼠骨组织中miR-16-5p表达明显降低,LRP6 mRNA表达明显升高。与A组相比,B组骨组织中LRP6、β-catenin及Runx2表达减低,骨体积分数、骨小梁厚度及生物力学性能减低。与A组相比,C组显著提高了骨组织中LRP6、β-catenin及Runx2的表达,骨体积分数、骨小梁厚度及生物力学性能均提高。与C组相比,D组结果显示LRP6、β-catenin及Runx2表达水平、骨量及生物力学性能均降低。结论 miR-16-5p能够通过靶向下调Wnt/β-catenin通路共受体中LRP6蛋白的表达而降低Wnt/β-catenin通路活性,降低小鼠骨量及骨生物力学性能,而力学载荷能够降低小鼠骨组织中miR-16-5p水平而提高LRP6蛋白表达及成骨活性和骨量。

Objective To explore whether LRP6 is a target gene of miR-16-5p and the role of miR-16-5p regulating the activity of Wnt/β-catenin pathway in promoting bone formation in mice under mechanical loading.Methods The target genes of miR-16-5p were predicted by microRNA information database, and luciferase reporter test was performed to verify whether LRP6 was the target gene of miR-16-5p.The treadmill group and the control group were constructed to verify the differential expression of miR-16-5p and LRP6 in bone tissue under different mechanical environments.The animal models of differential miR-16-5p expression and mechanical loading were made: Normal+miRNA Agomir control group(group A),Normal+miR-16-5p Agomir group(group B),Treadmill+miRNA Agomir Control group(group C) and Treadmill+miR-16-5p Agomir group(group D).After the intervention, the mice were killed to obtain bone tissue, and the changes of LRP6,β-catenin and Runx2 mRNA and protein expression in each group were detected.The changes of bone mass and mechanical properties were detected by Micro-CT scan of distal femur and biomechanical test of femoral shaft.Results microRNA database showed that LRP6 was the possible target gene of miR-16-5p, and luciferase report experiment confirmed that LRP6 was the target gene of miR-16-5p.Compared with the control group, the expression of miR-16-5p was significantly decreased and the expression of LRP6 mRNA was significantly increased in the treadmill group.Compared with group A,the expression of LRP6,β-catenin and Runx2 in bone tissue of group B decreased, and bone volume fraction, trabecular thickness and biomechanical properties decreased.Compared with group A,group C significantly increased the expression of LRP6,β-catenin and Runx2 in bone tissue, and bone volume fraction, trabecular thickness and biomechanical properties all increased.Compared with group C,the results of group D showed that the expression of LRP6,β-catenin and Runx2 decreased, and bone mass and biomechanical properties decreased.Conclusion miR-16-5p can down-regulate the expression of LRP6 protein and decrease the Wnt/β-catenin pathway activity, bone mass and bone biomechanical properties in mice, while mechanical loading can decrease the level of miR-16-5p and increase LRP6 protein expression and osteogenic activity and bone mass in mice.