MicroRNA-675-5p对成牙骨质细胞分化的影响
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摘要
目的:通过过表达或抑制microRNA-675-5p(miR-675-5p),观察miR-675-5p对小鼠成牙骨质细胞OCCM-30分化的影响。方法:用矿化诱导培养基诱导OCCM-30细胞分化,检测各矿化指标及miR-675-5p的含量变化。而后利用Lipofectamine 3000脂质体分别将miR-675-5p的mimic和inhibitor及对应的negative control转染进OCCM-30细胞,采用real-time PCR技术验证转染是否成功,继而采用real-time PCR和Western Blot的方法检测矿化相关指标ALP,OSX,RUNX2,BSP的含量变化,利用ALP活性试剂盒检测碱性磷酸酶活性变化。结果:Real-time PCR结果显示在OCCM-30细胞分化过程中miR-675-5p表达下调,在mimic组miR-675-5p表达上调明显,同时ALP,OSX,RUNX2表达下降,Western Blot证明OSX,RUNX2与BSP在蛋白水平上表达同样明显降低,ALP活性试剂盒检测提示ALP活性被抑制。Inhibitor组结果与mimic组恰好相反。结论:miR-675-5p抑制OCCM-30细胞的分化能力。
Objective: To investigate the effects of microRNA-675-5 p(miR-675-5 p) on cementoblast differentiation.Methods: The expression of miR-675-5 p was tested by real-time PCR during cementoblast differentiation of murine cementoblast cell line OCCM-30. Cells were then transfected with miR-675-5 p mimics/inhibitors or the mimic/inhibitor negative control using Lipofectamine 3000 Reagent. Real-time PCR was used to examine the expression level of miR-675-5 p after the transfection. The protein levels of RUNX2, OSX and BSP were determined by Western Blot. In addition, a kit of ALP activity assay was used to determine the activity of alkaline phosphatase.Results: The expression of miR-675-5 p was decreased during the cementoblast differentiation of OCCM-30 cells. Over-expression of miR-675-5 p in the mimic group decreased the protein levels of RUNX2, OSX and BSP and inhibited ALP activity. Besides, knock-down of miR-675-5 p increased the protein levels of RUNX2, OSX and BSP and promoted ALP activity.Conclusion: miR-675-5 p inhibits the ability of cementoblast differ-entiation and mineralization.
Objective: To investigate the effects of microRNA-675-5 p(miR-675-5 p) on cementoblast differentiation.Methods: The expression of miR-675-5 p was tested by real-time PCR during cementoblast differentiation of murine cementoblast cell line OCCM-30. Cells were then transfected with miR-675-5 p mimics/inhibitors or the mimic/inhibitor negative control using Lipofectamine 3000 Reagent. Real-time PCR was used to examine the expression level of miR-675-5 p after the transfection. The protein levels of RUNX2, OSX and BSP were determined by Western Blot. In addition, a kit of ALP activity assay was used to determine the activity of alkaline phosphatase.Results: The expression of miR-675-5 p was decreased during the cementoblast differentiation of OCCM-30 cells. Over-expression of miR-675-5 p in the mimic group decreased the protein levels of RUNX2, OSX and BSP and inhibited ALP activity. Besides, knock-down of miR-675-5 p increased the protein levels of RUNX2, OSX and BSP and promoted ALP activity.Conclusion: miR-675-5 p inhibits the ability of cementoblast differ-entiation and mineralization.
引文
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[2] Wang C, Liao H, Sun H, et al. MicroRNA-3064-3p regulates the differentiation of cementoblasts through targeting DKK1[J]. J Periodontal Res, 2018,53(5):705-713.
[3] Wang X, Sun H, Liao H, et al. MicroRNA-155-3p mediates TNF-alpha-inhibited cementoblast differentia-tion[J]. J Dental Res, 2017,96(12):1 430-1 437.
[4] Liang WC, Fu WM, Wang YB, et al. H19 activates Wnt signaling and promotes osteoblast differentiation by functioning as a competing endogenous RNA[J]. Sci Rep, 2016,6:20121.
[5] Cao ZG, Zhang H, Zhou X, et al. Genetic evidence for the vital function of Osterix in cementogenesis[J]. J Bone Miner Res, 2012,27(5):1 080-1 092.
[6] Huang YP, Zheng YF, Jia LF, et al. Long noncoding RNA H19 promotes osteoblast differentiation via TGF-beta1/Smad3/HDAC signaling pathway by deriving miR-675[J]. Stem Cells, 2015,33(12):3 481-3 492.
[7]吴浩,韩红娟,任小华,等.缺氧环境对成牙骨质细胞成骨功能调节的研究[J].成都医学院学报,2015,10(2):152-154.Wu H, Han HJ, Ren XH, et al. Effect of hypoxia on osteoblast function of cementoblast-like cell[J]. Journal of Chengdu Medical College, 2015,10(2):152-154.
[8] Konstantonis D, Papadopoulou A, Makou M, et al.The role of cellular senescence on the cyclic stretching-mediated activation of MAPK and ALP expression and activity in human periodontal ligament fibroblasts[J].Exp Gerontol, 2014,57(9):175-180.