Exosomes derived from circRNA Rtn4-modified BMSCs attenuate TNF-α-induced cytotoxicity and apoptosis in murine MC3T3-E1 cells by sponging miR-146a
Exosomes derived from circRNA Rtn4-modified BMSCs attenuate TNF-α-induced cytotoxicity and apoptosis in murine MC3T3-E1 cells by sponging miR-146a
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DOI：
10.1042/bsr20193436
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发表时间：
2020-05-26
影响因子：
4
通讯作者：
Li, Jinhua
中科院分区：
文献类型：
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作者：
Cao, Guijun;Meng, Xianqing;Li, Jinhua
骨质疏松症是世界范围内最常见和最复杂的骨骼疾病。骨髓间充质干细胞（BMSCs）分泌的外泌体被认为是骨组织再生的理想种子源。然而，骨髓间充质干细胞分泌的外泌体（BMSCs-Exos）在骨质疏松症中的作用及其机制尚不清楚。在本研究中，microRNA（miRNA）-146a和circular RNA（circRNA）Rtn 4（circ-Rtn 4）的表达通过定量实时聚合酶链反应（qRT-PCR）进行了评估，并通过蛋白质印迹法测定其蛋白质表达。酶联免疫吸附法检测caspase-3活性。分别采用MTT法和流式细胞仪分析法检测细胞活力和细胞凋亡。荧光素酶报告基因测定用于靶标验证。结果显示，肿瘤坏死因子-α（TNF-α）剂量依赖性地增加miR-146 a表达，抑制细胞活力，并促进细胞凋亡，如增加的caspase-3，裂解的caspase-3和Bcl-2相关的X蛋白（Bax）表达以及caspase-3活性所示。然而，miR-146 a沉默或与BMSCs-Exos共培养阻断了这些作用。此外，与来源于circ-Rtn 4修饰的BMSC的exosomes（Rtn 4-Exos）共培养减弱了TNF-α诱导的MC 3 T3-E1细胞的细胞毒性和凋亡，如通过caspase-3、切割的caspase-3和Bax蛋白表达和caspase-3活性的降低所证明的。此外，miR-146 a被鉴定为circ-Rtn 4的靶点，并且Rtn 4-Exos通过海绵状吸收miR-146 a而在TNF-α处理的MC 3 T3-E1细胞中发挥其功能。因此，我们的研究结果表明，Rtn 4-Exos减弱TNF-α诱导的细胞毒性和凋亡的小鼠MC 3 T3-E1细胞中的海绵状的miR-146 a，这表明Rtn 4-Exos可能作为新的候选人治疗骨质疏松症。
Osteoporosis is the most common and complex skeletal disorder worldwide. Exosomes secreted by bone marrow-derived mesenchymal stromal cells (BMSCs) are considered as an ideal seed source for bone tissue regeneration. However, the role of exosomes secreted by BMSCs (BMSCs-Exos) in osteoporosis and its underlying mechanisms remain unclear. In the present study, the expression of microRNA (miRNA)-146a and circular RNA (circRNA) Rtn4 (circ-Rtn4) was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), and their protein expression was determined by Western blotting. Enzyme-linked immunosorbent assay was performed to detect caspase-3 activity. Cell viability and apoptosis were assessed using 3-(4,5-Dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry analysis, respectively. Luciferase reporter assay was exploited for target validation. Results showed that tumor necrosis factor-alpha (TNF-alpha) dose-dependently increased miR-146a expression, inhibited cell viability, and promoted cell apoptosis, as indicated by increased caspase-3, cleaved caspase-3, and Bcl-2-associated X protein (Bax) expression as well as caspase-3 activity. However, miR-146a silencing or co-culture with BMSCs-Exos blocked these effects. Moreover, co-culture with exosomes-derived from circ-Rtn4-modified BMSCs (Rtn4-Exos) attenuated TNF-alpha-induced cytotoxicity and apoptosis inMC3T3-E1 cells, as evidenced by the decrease in caspase-3, cleaved caspase-3, and Bax protein expression and caspase-3 activity. In addition, miR-146a was identified as a target of circ-Rtn4, and Rtn4-Exos exerted their function in TNF-alpha-treated MC3T3-E1 cells by sponging miR-146a. Hence, our findings suggested that Rtn4-Exos attenuated TNF-alpha-induced cytotoxicity and apoptosis in murine MC3T3-E1 cells by sponging miR-146a, suggesting that Rtn4-Exos may serve as novel candidates for treating osteoporosis.