微小RNA-218-5p靶向Jagged 1调控人牙周膜干细胞的活性和骨向分化

MicroRNA-218-5p regulates the activity and osteogenic differentiation of human periodontal ligament stem cells by targeting Jagged 1

目的研究微小RNA-218-5p(miR-218-5p)对人牙周膜干细胞(hPDLSCs)活性和骨向分化的影响并探讨其机制,为牙周炎的治疗提供研究基础。方法将anti-miR-218-5p组(转染anti-miR-218-5p)、anti-miR-NC组(转染anti-miR-NC)、pcDNA组(转染pcDNA)、pcDNA-JAG1组(转染pcDNA-JAG1)、anti-miR-218-5p+si-NC组(共转染anti-miR-218-5p和si-NC)、anti-miR-218-5p+si-JAG1组(共转染anti-miR-218-5p和si-JAG1),用脂质体法转染至hPDLSCs细胞;运用实时荧光定量逆转录聚合酶链反应(qRT-PCR)法检测细胞中miR-218-5p、Ⅰ型胶原蛋白(Col-1)、骨钙素、Runt相关转录因子2(Runx2)的表达;蛋白质印迹法(Westrn blotting)检测细胞中Jagged 1(JAG1)的蛋白表达;MTT法检测细胞活性;茜素红染色实验检测细胞的矿化结节;双荧光素酶报告基因检测实验检测细胞的荧光活性。结果与anti-miR-NC组相比,anti-miR-218-5p组hPDLSCs细胞培养48、72 h时,细胞活性升高[48 h:(0.44±0.04)比(0.62±0.06);72 h:(0.53±0.05)比(0.83±0.08);P<0.001],细胞的矿化结节明显升高,Col-1[(0.26±0.03)比(0.74±0.07)]、骨钙素[(0.21±0.02)比(0.54±0.05)]、Runx-2[(0.29±0.03)比(0.61±0.06)]蛋白相对表达量均显著升高(均P<0.001)。与pcDNA组相比,pcDNA-JAG1组hPDLSCs细胞培养48、72 h时,细胞活性显著升高[48 h:(0.42±0.04)比(0.59±0.05);72 h:(0.55±0.05)比(0.81±0.08);P<0.001],Col-1[(0.34±0.03)比(0.71±0.07)]、骨钙素[(0.23±0.02)比(0.48±0.04)]、Runx-2[(0.25±0.03)比(0.56±0.05)]蛋白表达量均显著升高(均P<0.001)。miR-218-5p可靶向调控JAG1的表达。抑制JAG1可逆转抑制miR-218-5p对hPDLSCs的活性和骨向分化作用。结论抑制miR-218-5p可促进人牙周膜干细胞活性和骨向分化,其机制与靶向JAG1有关。

Objective To investigate the effect of microRNA-218-5p(miR-218-5p)on the activity and osteogenic differentiation of human periodontal ligament stem cells(hPDLSCs)and to explore its mechanism,and to provide research basis for the treatment of peri-odontitis.Methods Cells were divided into anti-miR-218-5p group(transfected with anti-miR-218-5p),anti-miR-NC group(transfect-ed with anti-miR-NC),pcDNA group(transfected with pcDNA),pcDNA-JAG1 group(transfected with pcDNA-JAG1),anti-miR-218-5p+si-NC group(co-transfected with anti-miR-218-5p and si-NC),anti-miR-218-5p+si-JAG1 group(co-transfected with anti-miR-218-5p and si-JAG1),transfected into hPDLSCs cells by liposome method.Real-time fluorescence quantitative reverse transcription poly-merase chain reaction(qRT-PCR)method was used to detect miR-218-5p,type I collagen(Col-1),osteocalcin(OCN),Runt-related tran-scription factor 2(Runx2)in cells;Western blotting method was used to detect the protein expression of Jagged 1(JAG1)in cells;tetra-methylazolylazolyl salt microenzyme reaction colorimetric method(MTT)was used to detect cell viability;alizarin red staining test was used to detect mineralized nodules of cells;dual luciferase reporter assay was used to detect the fluorescence activity of cells.Results Compared with the anti-miR-NC group,the cell viability of hPDLSCs in the anti-miR-218-5p group was increased at 48 and 72 h[48 h:(0.44±0.04),(0.62±0.06);72 h:(0.53±0.05),(0.83±0.08);P<0.001];the mineralized nodules of cells were significantly increased,and the relative expressions of Col-1,OCN,and Runx-2 proteins were significantly increased[Col-1 protein:(0.26±0.03),(0.74±0.07);OCN protein:(0.21±0.02),(0.54±0.05);Runx-2 protein:(0.29±0.03),(0.61±0.06);P<0.001].Compared with the pcDNA group,the cell via-bility of hPDLSCs in the pcDNA-JAG1 group was significantly increased when cultured for 48 and 72 h[48 h:(0.42±0.04),(0.59±0.05);72 h:(0.55±0.05),(0.81±0.08);P<0.001],the expression of Col-1,OCN,and Runx-2 proteins all were increased significantly[Col-1 protein:(0.34±0.03),(0.71±0.07);OCN protein:(0.23±0.02),(0.48±0.04);Runx-2 protein:(0.25±0.03),(0.56±0.05);P<0.001].miR-218-5p can target the expression of JAG1.Inhibition of JAG1 reversibly inhibited the activity and osteogenic differentiation of hP-DLSCs by miR-218-5p.Conclusions Inhibition of miR-218-5p can promote the activity and osteogenic differentiation of human peri-odontal ligament stem cells.The mechanism is related to the targeting of JAG1.