miR-508-3p靶向AFF4/TGFβ1信号通路调控人牙髓细胞分化的机制研究

The mechanism of miR-508-3p on the regulation of the differentiation of human dental pulp cellsby targeting AFF4/TGFβ1 signaling pathway

目的:探讨miR-508-3p对缺氧牙髓细胞分化的影响及其机制。方法:用氯化钴(CoCl2)诱导缺氧模型,记为实验组。用等量生理盐水处理的牙髓细胞为对照组。脂质体法将miR-NC、miR-508-3p、si-NC、si-AFF4、miR-508-3p+pcDNA、miR-508-3p+pcDNA-AFF4转染缺氧牙髓细胞。RT-qPCR法检测miR-508-3p、AFF4的表达及分化相关基因牙本质基质蛋白(DMP1)、牙本质涎磷蛋白(DSPP)、骨钙素(OCN)、碱性磷酸酶(ALP)的表达。Western blotting法检测细胞中AFF4、DMP1、DSPP、OCN、ALP、转化生长因子β1(TGFβ1)、Smad、骨形态发生蛋白(BMP)的表达。双荧光素酶报告实验检测miR-508-3p与AFF4的靶向关系。结果:与对照组相比,实验组牙髓细胞miR-508-3p表达显著升高,AFF4、DMP1、DSPP、OCN、ALP的表达均显著降低(均P<0.01)。过表达miR-508-3p可显著抑制DMP1、DSPP、OCN、ALP的表达,敲减AFF4具有相似的作用(均P<0.01)。双荧光素酶报告实验显示,miR-508-3p与AFF4存在互补的结合位点,且过表达AFF4逆转了过表达miR-508-3p对缺氧牙髓细胞的分化抑制作用(P<0.01)。上调miR-508-3p表达或下调AFF4表达能显著降低TGFβ1、Smad、BMP蛋白表达(P<0.01)。结论:miR-508-3p通过靶向AFF4抑制下游TGFβ1信号通路的活性发挥抑制缺氧牙髓细胞分化作用。

Objective: To investigate the effect and mechanism of miR-508-3 p on the differentiation of hypoxic dental pulp cells(DPCs). Methods: The DPCs were treated with cobalt chloride(CoCl2), a hypoxia-mimicking agent, to establish a cellular model of hypoxic DPCs(experimental group). The DPCs treated with normal saline were defined asa control group. The hypoxic DPCs were transfected with miR-NC, miR-508-3 p, si-NC, si-AFF4,miR-508-3 p+pcDNA, ormiR-508-3 p+pcDNA-AFF4 by using the liposome method. RT-qPCR was used to detect the expressions of miR-508-3 p, AFF4, dentin matrix protein 1(DMP1), dentin sialophosphoprotein(DSPP), osteocalcin(OCN), and alkaline phosphatase(ALP). Western blotting was used to detect the protein expressions of AFF4, DMP1, DSPP, OCN, ALP, transforming growth factor β1(TGFβ1), Smad, and bone morphogenetic protein(BMP). Double-Luciferase reporter assay was used to confirm the targeting relationship between miR-508-3 p and AFF4. Results: Compared with the control group, the miR-508-3 p expression was significantly upregulated,while the expressions of AFF4, DMP1, DSPP, OCN, and ALP were downregulated in the experimental group(P<0.01). Overexpression of miR-508-3 p significantly inhibited the expression of AFF4, DMP1, DSPP, OCN,and ALP. The effect of AFF4 knockdown was similar to that of miR-508-3 p overexpression(P<0.01). DoubleLuciferase reporter assay showed that miR-508-3 p targeted the 3’-UTR of AFF4. Overexpression of AFF4 reversed the inhibitory effect of miR-508-3 p on hypoxic DPCs differentiation(P<0.01). Upregulation of miR-508-3 p or downregulation of AFF4 significantly reduced the expression levels of TGFβ1, Smad and BMP protein(P<0.01). Conclusion: miR-508-3 p inhibits the differentiation of hypoxic DPCs by targeting AFF4 and inhibiting downstream TGFβ1 signaling pathway.