模拟失重下miR-138-5p靶向SIRT1负调控成骨细胞分化的研究

徐丽群; 张丽君; 李高志; 张晓雁; 王艺璇; 胡泽兵; 曹新生; 石菲; 张舒

doi:10.3969/j.issn.2095-5227.2021.10.014

模拟失重下miR-138-5p靶向SIRT1负调控成骨细胞分化的研究

miR-138-5p inhibits osteoblast differentiation by downregulating SIRT1 expression under simulated weightlessness

摘要

摘要:
背景机械刺激对于维持骨骼重塑和平衡至关重要，机械卸载引起的骨骼系统损伤是长期航天飞行的主要局限性之一。研究表明微小RNA(miRNA)可通过调控与骨形成相关的基因和信号通路调节成骨细胞功能。
目的探究模拟失重下miR-138-5p/SIRT1信号通路对成骨细胞分化的影响，为失重性骨质丢失的在体靶向干预寻求治疗靶点。
方法利用2D回转器对前成骨细胞MC3T3-E1进行模拟失重处理，验证沉默信息调节因子2相关酶I(SIRT1)及miR-138-5p在模拟失重下的表达发生变化；向MC3T3-E1细胞中转染miR-138-5p mimic、inhibitor、pcDNA3.1(+)-SIRT1及相应的阴性对照进行功能验证；向MC3T3-E1细胞中转染inhibitor -NC、miR-138-5p inhibitor后模拟失重处理48 h进行挽救实验；采用qRT-PCR方法检测miRNA及SIRT1、Runx2、Osx的mRNA表达变化；采用Western blot方法检测SIRT1、Runx2、Osx的蛋白表达变化；采用碱性磷酸酶(alkaline phosphatase，ALP)活性检测和ALP染色方法检测成骨细胞分化能力的变化。
结果模拟失重下，MC3T3-E1细胞中Runx2、SIRT1的mRNA及蛋白均表达下降(P＜0.01或P＜0.05)；转染miR-138-5p mimic和inhibitor后，qRT-PCR、Western blot、ALP活性及ALP染色检测均表明过表达miR-138-5p能够显著抑制成骨细胞分化，而抑制miR-138-5p能够促进MC3T3-E1细胞分化(P＜0.05或P＜0.01)。miR-138-5p mimic与pEX-SIRT1共转染实验表明miR-138-5p通过抑制SIRT1负调控成骨细胞分化(P＜0.01)。此外，抑制miR-138-5p可以降低模拟失重对成骨细胞的分化抑制，表现为促进SIRT1、Runx2、Osx mRNA和蛋白表达(P＜0.01)。
结论 SIRT1表达在模拟失重下下调，而miR-138-5p表达上调；miR-138-5p通过直接靶向SIRT1调控Runx2、Osx的mRNA及蛋白表达，抑制成骨细胞分化；此外，抑制miR-138-5p/SIRT1通路可以部分降低模拟失重对成骨细胞的分化抑制，miR-138-5p可以作为潜在的干预靶点。

Abstract:
Background Mechanical stimulation plays a vital role in maintaining bone remodeling and homeostasis, and mechanical unloading-induced skeletal system damage is one of the main limitations for long-term space flight. miRNA and SIRT1 can regulate osteoblasts functions via regulating signaling pathways related to bone formation.
Objective To explore the effect of miR-138-5p/SIRT1 signaling pathway on osteoblast differentiation under simulated weightlessness condition, and provide a therapeutic target for intervention of weightlessness-induced bone loss.
Methods 2D clinostat was used to culture MC3T3-E1 under simulated weightlessness to verify the changes of expression of SIRT1. miR-138-5p mimic, miR-138-5p inhibitor, pcDNA3.1(+)-SIRT1 and the corresponding negative control were transfected into MC3T3-E1 cells for functional experiments, MC3T3-E1 cells were treated with simulated weightlessness for 48h after transfecting with inhibitor-NC or miR-138-5p inhibitor, and qRT-PCR was used to detect the mRNA expressions of miRNA and SIRT1, Runx2, Osx mRNA. The protein expression of SIRT1, Runx2 and Osx were detected by Western blot, and Alkaline phosphatase (ALP) activity and ALP staining were used to detect osteoblast differentiation.
Results The expressions of Runx2, SIRT1 mRNA and protein in MC3T3-E1 cells decreased under simulated weightlessness (P＜0.01 or P＜0.05); After transfecting miR-138-5p mimic or inhibitor into MC3T3-E1 cells, qRT-PCR, Western Blot, ALP activity and ALP staining detection showed that miR-138-5p significantly inhibited osteoblast differentiation (P＜0.01 or P＜0.05). After miR-138-5p mimic and pEX-SIRT1 being co-transfecting into MC3T3-E1 cells, the result showed that miR-138-5p negatively regulated osteoblast differentiation via inhibiting SIRT1 (P＜0.01). Furthermore, knockdown of endogenous miR-138-5p could alleviate the suppression of SIRT1 expression and osteoblastic differentiation under simulated weightlessness (P＜0.01).
Conclusion SIRT1 is downregulated while miR-138-5p is upregulated under simulated weightlessness. miR-138-5p regulates the protein expression level of Runx2 and Osx via targeting SIRT1 and inhibits osteoblast differentiation. In addition, silencing miR-138-5p/SIRT1 alleviates the suppression effect of simulated weightlessness on osteoblast differentiation, indicating miR-138-5p may be a potential target for weightlessness-induced bone loss.

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