miRNA-222靶向Smad2/7对牙周膜干细胞向成骨分化的影响

Effect of miRNA-222 Targeting Smad2/7 on Osteogenic Differentiation of Periodontal Ligament Stem Cells

目的:探讨miRNA-222靶向Smad2/7对牙周膜干细胞向成骨分化的影响。方法:采用流式细胞术鉴定人牙周膜干细胞表面标志物,采用油红O染色、茜素红染色和阿尔新蓝染色法验证成脂、成骨和软骨分化能力,同时验证miRNA-222、sh-Smad2和sh-Smad7对成骨分化的影响,RT-PCR检测miRNA-222在成骨分化中的表达,采用Western blot检测miRNA-222对ALP、Runx2、OCN、Smad2和Smad7蛋白的影响。结果:牙周膜干细胞表面呈阳性表达的有STRO1和CD146,阳性率分别为98.63%和99.16%。RT-PCR检测结果显示,在成骨分化过程中,人牙周膜干细胞中miRNA-222表达明显降低。茜素红染色结果显示,与空白组相比,miRNA-222模拟组中的红褐色矿化结节的数量明显降低(P<0.05),miRNA-222抑制组中的红褐色矿化结节的数量显著增多(P<0.05);与sh-NC相比,sh-Smad2组和sh-Smad7组红褐色矿化结节的数量显著减少(P<0.05)。Western blot检测结果显示,与空白组相比,miRNA-222模拟组的ALP、Runx2、OCN蛋白表达明显降低(P<0.05),与miRNA-222模拟组相比,miRNA-222抑制组的ALP、Runx2、OCN蛋白表达显著升高(P<0.05);与空白组相比,miRNA-222模拟组的Smad2和Smad7蛋白表达明显降低(P<0.05),与miRNA-222模拟组相比,miRNA-222抑制组的Smad2和Smad7蛋白表达显著升高(P<0.001)。结论:过表达miRNA-222可以减少Smad2和Smad7表达,从而抑制人牙周膜干细胞的成骨分化。

Objective To investigate the effect of miRNA-222 targeting Smad2/7 on the osteogenic differentiation of periodontal ligament stem cells (PDLSCs).Methods Flow cytometry was used to identify the surface markers of human periodontal ligament stem cells.Oil red O staining,alizarin red staining and alcian blue staining were used to verify the adipogenic,osteogenic and chondrogenic differentiation abilities.The effects of miRNA-222,sh-Smad2 and sh-Smad7 on osteogenic differentiation were also verified.The expression of miRNA-222 in osteogenic differentiation was detected by RT-PCR,Western blot was used to detect the effect of miRNA222 on the expression of ALP,Runx2,OCN,Smad2 and Smad7 proteins.Results STRO1 and CD146 were positively expressed on the surface of periodontal ligament stem cells,and the positive rates were 98.63% and 99.16%,respectively.RT-PCR results showed that during the process of osteogenic differentiation,the expression of miRNA-222 in human periodontal ligament stem cells was significantly reduced.The results of Alizarin Red staining showed that compared with the blank group,the number of red-brown mineralized nodules in the miRNA-222 simulation group was significantly reduced (P<0.05),and the red-brown mineralized nodules in the miRNA-222 inhibition group were significantly reduced.The number was significantly increased (P<0.05),compared with sh-NC,the number of red-brown mineralized nodules in the sh-Smad2 and sh-Smad7 groups was significantly reduced (P<0.05).Western blot detection results showed that compared with the blank group,the expression of ALP,Runx2,and OCN in the miRNA-222 simulation group was significantly reduced (P<0.05).Compared with the miRNA-222 simulation group,the ALP,Runx2,and OCN protein expressions in the miRNA-222 suppression group The protein expression of Runx2 and OCN was significantly increased (P<0.05),compared with the blank group,the expression of Smad2 and Smad7 protein in the miRNA-222 simulation group was significantly reduced (P<0.05).Compared with the miRNA-222 simulation group,the expression of The protein expression of Smad2 and Smad7 in the miRNA-222 inhibition group was significantly increased (P<0.001).Conclusion Overexpression of miRNA-222 can reduce the expression of Smad2 and Smad7,thereby inhibiting the osteogenic differentiation of human periodontal ligament stem cells.