circSEC31A调控miR-324-5p对脂肪干细胞增殖、凋亡和成骨分化的影响

Effect of CircSEC31A Regulating MiR-324-5p on Adipose Stem Cell Proliferation,Apoptosis and Osteogenic Differentiation

目的探讨环状非编码RNA SEC31A(circSEC31A,又名hsa_circ_0006618)对脂肪干细胞增殖、凋亡和成骨分化的影响和机制。方法在脂肪干细胞中分别转染sh-circSEC31A和LV-circSEC31A后,细胞增殖采用CCK8法检测;细胞凋亡采用流式细胞仪检测;circSEC31A和miR-324-5p靶点结合情况采用双荧光素酶测定法检测;成骨分化相关分子RUNX2和OPN的mRNA和蛋白水平分别采用Western blot和RT-qPCR检测。结果在脂肪干细胞中转染sh-circSEC31A后能够明显抑制细胞的增殖和成骨分化相关分子RUNX2、OPN的表达水平(P<0.05),促进细胞的凋亡(P<0.05)。在脂肪干细胞中转染LV-circSEC31A后能够明显促进细胞的增殖和成骨分化相关分子RUNX2、OPN的表达水平(P<0.05),抑制细胞的凋亡(P<0.05)。双荧光素酶结果显示circSEC31A能够靶向结合miR-324-5p(P<0.05)。回复实验显示,在脂肪干细胞中共转染LV-circSEC31A和miR-324-5p mimics后能够反转单独转染LV-circSEC31A对脂肪干细胞增殖、凋亡和成骨分化的影响;共转染LV-circSEC31A和miR-324-5p inhibitors后能够进一步加强单独转染LV-circSEC31A对脂肪干细胞增殖、凋亡和成骨分化的影响。结论circSEC31A能够通过靶向调控miR-324-5p参与脂肪干细胞增殖、凋亡和成骨分化,circSEC31A可能成为干预脂肪干细胞生物学功能的靶点。

Objective To explore the effect and mechanism of circular non-coding RNA SEC31A(circSEC31A,also known as hsa_circ_0006618)on proliferation,apoptosis and osteogenic differentiation of adipose stem cells.Methods After transfection of sh-circSEC31A and LV-circSEC31A respectively,cell proliferation was detected by CCK8 method,apoptosis by flow cytometry,the binding of circSEC31A and miR-324-5p by dual luciferase assay.Western blot and RT-qPCR were used to detect mRNA and protein levels of RUNX2 and OPN,which were related to osteogenic differentiation respectively.Results Transfection of sh-circSEC31A could significantly inhibit the expression levels of RUNX2 and OPN,which were related to cell proliferation and osteogenic differentiation(P<0.05),and promote cell apoptosis(P<0.05).Transfection of LV-circSEC31A in adipocytes could significantly promote the expression levels of RUNX2 and OPN related molecules in cell proliferation and osteogenic differentiation(P<0.05),and inhibit cell apoptosis(P<0.05).The dual luciferase results showed that circSEC31A could target bind miR-324-5p(P<0.05).The recovery experiment showed that the effects of single transfection of LV-circSEC31A and miR-324-5p Mimics on the proliferation,apoptosis and osteogenic differentiation of adipocytes could be reversed.After co-transfection of LV-circSEC31A and miR-324-5p inhibitors,the influence of single transfection of LV-circSEC31A on proliferation,apoptosis and osteogenic differentiation of adipose stem cells could be further enhanced.Conclusion CircSEC31A can be involved in adipocyte proliferation,apoptosis and osteogenic differentiation by targeting miR-324-5p,and may be a target for intervention of adipocyte biological function.