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| miR-381-3p通过靶向ERK1/2/ETS1信号通路影响MC3T3-E1细胞成骨分化 |
| miR-381-3p affects the osteogenic differentiation of MC3T3-E1 cells by targeting the ERK1/2/ETS1 signaling pathway |
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| DOI：10.3969/j.issn.1006.7108.2022.03.003 |
| 中文关键词: 微小RNA 骨质疏松 MC3T3-E1细胞 细胞外信号调节激酶 作用机制 成骨分化 |
| 英文关键词:microRNA osteoporosis MC3T3-E1 cells extracellular signal-regulated kinase mechanism of action osteogenic differentiation |
| 基金项目:福建省自然科学基金面上项目(2019J01173)；2019福建省卫生教育联合攻关项目（2019-WJ-01）；卫生健康委医学创新课题（2019-CX-1） |
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| 中文摘要: |
| 目的 探讨miR-381-3p对MC3T3-E1细胞成骨分化的影响和作用机制。方法 慢病毒转染细胞后实验分为NC mimic miR-381-3p组、mimic miR-381-3p组、NC inhibitor miR-381-3p组和inhibitor miR-381-3p组；运用实时荧光定量PCR（qRT-PCR）法检测不同组miR-381-3p的表达水平以验证转染效率；诱导MC3T3-E1细胞成骨分化后，检测各组细胞中碱性磷酸酶 (ALP) 活性水平；茜素红染色法检测各组细胞的成骨矿化能力；qRT-PCR测定各组细胞中ALP、Runx2、PPARγ和ETS1 mRNA表达水平；蛋白免疫印迹法测定各组细胞中collagen Ⅰ、ALP、Runx2、PPARγ、ETS1、P-ERK1/2水平；并用MAPK/ERK通路抑制剂（PD98059）干预各组细胞后进行目的蛋白检测。结果 与NC mimic miR-381-3p组、mimic miR-381-3p组及NC inhibitor miR-381-3p组比较，inhibitor miR-381-3p组中细胞中ALP、Runx2、ETS1 mRNA的水平均明显升高，并且collagen Ⅰ、ALP、Runx2、ETS1、P-ERK1/2蛋白的表达水平也明显升高，但是PPARγ mRNA和蛋白表达水平是下调的；与其他3组对比，inhibitor miR-381-3p组中细胞的ALP活性和钙化能力检测显示均显著高表达。MAPK通路抑制后蛋白实验结果也佐证了miR-381-3p参与调控MC3T3-E1细胞的成骨分化过程。结论 miR-381-3p通过下调ERK1/2/ETS1信号通路表达水平进而抑制了MC3T3-E1细胞的成骨分化能力。 |
| 英文摘要: |
| Objective To explore the effect and mechanism of miR-381-3p on the osteogenic differentiation of MC3T3-E1 cells. Methods The lentivirus-transfected cells were divided into NC mimic miR-381-3p group, mimic miR-381-3p group, and NC inhibitor miR-381-3p group and inhibitor miR-381-3p group. Real-time fluorescent quantitative PCR (qRT-PCR) method was used to detect the expression levels of miR-381-3p in different groups to verify the transfection efficiency. After inducing osteogenic differentiation of MC3T3-E1 cells, the levels of alkaline phosphatase (ALP) activity in the cells of each group were detected. Alizarin red staining was used to detect the osteogenic mineralization ability of each group of cells. qRT-PCR was used to determine the expression levels of ALP, Runx2, PPARγ, and ETS1 mRNA in each group of cells. Western blotting was used to determine the protein levels of collagen Ⅰ, ALP, Runx2, PPARγ, ETS1, and P-ERK1/2 in each group of cells. MAPK/ERK pathway inhibitor (PD98059) was used to intervene in each group of cells to detect the target protein. Results Compared to those in NC mimic miR-381-3p group, mimic miR-381-3p group, and NC inhibitor miR-381-3p group, the levels of ALP, Runx2, and ETS1 mRNA in the cells of inhibitor miR-381-3p group increased significantly. The expression levels of collagen Ⅰ, ALP, Runx2, ETS1, and P-ERK1/2 protein also significantly increased, but the expression levels of PPARγ mRNA and protein were down-regulated. Compared to those in the other three groups, ALP activity and calcification ability of the cells in the inhibitor miR-381-3p group showed significantly high expression. The protein experiment results after the inhibition of the MAPK pathway also confirmed that miR-381-3p participated in the regulation of the osteogenic differentiation process of MC3T3-E1 cells. Conclusion miR-381-3p inhibits the osteogenic differentiation ability of MC3T3-E1 cells by down-regulating the expression level of ERK1/2/ETS1 signaling pathway. |
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