miR-24-3p靶向BMP8B对成骨细胞功能影响的实验研究

Experimental Study on the Effect of miR-24-3p Targeting BMP8B on the Function of Osteoblasts

目的:探讨miR-24-3p与骨形态发生蛋白8B(BMP8B)的靶向关系,并分析二者对成骨细胞功能的影响。方法:体外分离培养人牙周膜干细胞(hPDLSCs),并将其诱导分化为成骨细胞,采用碱性磷酸酶(ALP)染色法、茜素红染色法鉴定成骨细胞;将成骨细胞分为空白对照组、阴性对照组(inhibitor-NC组)、沉默miR-24-3p表达组(miR-24-3p-inhibitor组)、共转染组(miR-24-3p-inhibitor+BMP8B-siRNA组)。采用实时荧光定量PCR法检测miR-24-3p、BMP8B mRNA水平;CCK-8法、流式细胞仪分别检测各组成骨细胞增殖、凋亡情况;采用ALP活性检测试剂盒检测ALP活性;免疫印迹法检测各组成骨细胞BMP8B蛋白、功能活性相关蛋白(PICP、BGP、OPN)水平。应用TargetScan数据库预测miR-24-3p与BMP8B靶向关系并用双荧光素酶报告基因实验验证。结果:与空白对照组、inhibitor-NC组比较,miR-24-3p-inhibitor组成骨细胞增殖抑制率、凋亡率降低,ALP活性及PICP、BGP、OPN表达升高(P<0.05);与miR-24-3p-inhibitor组比较,miR-24-3p-inhibitor+BMP8B-siRNA组成骨细胞增殖抑制率、凋亡率升高,ALP活性及PICP、BGP、OPN表达降低(P<0.05)。双荧光素酶报告基因实验证实miR-24-3p靶向调控BMP8B。结论:抑制miR-24-3p可能通过靶向上调BMP8B表达促进成骨细胞增殖及分化,并抑制细胞凋亡。

Objective:To investigate the targeting relationship between miR-24-3p and bone morphogenetic protein 8B(BMP8B),and to analyze their effects on osteoblast function.Methods:Human periodontal ligament stem cells(hPDLSCs)were isolated and cultured in vitro and induced to differentiate into osteoblasts.The osteoblasts were identified by alkaline phosphatase(ALP)staining and alizarin red staining;the osteoblasts were divided into blank control group,negative control group(inhibitor-NC group),silencing miR-24-3p expression group(miR-24-3p-inhibitor group),and co-transfection group(miR-24-3p-inhibitor+BMP8B-siRNA group).Real-time fluorescent quantitative PCR method was used to detect the levels of miR-24-3p and BMP8B mRNA;CCK-8 method and flow cytometer were used to detect the proliferation and apoptosis of bone cells in each component;ALP activity detection kit was used to detect ALP activity;Western blotting was used to detect the levels of BMP8B protein and functional activity-related proteins(PICP,BGP,OPN)in each component of bone cells.The TargetScan database and the dual luciferase reporter gene experiment were used to predict and verify the targeting relationship between miR-24-3p and BMP8B respectively.Results:Compared with the blank control group and inhibitor-NC group,the osteoblast proliferation inhibition rate and apoptosis rate in the miR-24-3p-inhibitor group were decreased,ALP activity and expression of PICP,BGP,OPN were increased(P<0.05);compared with miR-24-3p-inhibitor group,the osteoblast proliferation inhibition rate and apoptosis rate in the miR-24-3p-inhibitor+BMP8B-siRNA group were increased,ALP activity and expression of PICP,BGP,OPN were decreased(P<0.05).The dual luciferase reporter gene experiment confirmed that miR-24-3p targeted regulation of BMP8B.Conclusion:Inhibition of miR-24-3p may promote the proliferation and differentiation of osteoblasts and inhibit cell apoptosis by targeting up-regulation of BMP8B expression.