miR-148a-3p靶向Wnt1基因对人牙髓干细胞增殖活力及成骨分化的影响.pdf

Chin J Prosthodont,September 2023,Vol.24,No.5口腔颌面修复学杂志2023年9月第24卷第5期 论著 miR-miR-148148a-a-3 3p p靶向靶向WntWnt1 1基因对人牙髓干细胞增殖活力及成骨分化的影响基因对人牙髓干细胞增殖活力及成骨分化的影响吴东朱青青高振杰【摘要】目的：观察微小RNA（miRNA）-148a-3p对人牙髓干细胞（DPSCs）增殖活力及成骨分化的影响，并探讨可能机制。方法：取对数期 DPSCs，分为空白组、NC 组、inhibitor 组、inhibitor+XAV939无翅型MMTV整合位点家族蛋白（Wnt）/-连环蛋白（-catenin）信号通路抑制剂组。空白组不处理，NC组转染阴性对照质粒miR-148a-3p NC，inhibitor组转染miR-148a-3p inhibitor，inhibitor+XAV939组转染miR-148a-3p inhibitor并加入XAV939（40 mol/L）。转染48 h后qRT-PCR法检测miR-148a-3p表达量；MTT法检测增殖活力；ELISA法检测碱性磷酸酶（ALP）活性；茜素红染色法检测矿化能力；双荧光素酶实验验证miR-148a-3p靶向基因；Western blot法检测糖原合成酶激酶3（GSK-3）、-catenin、Runt相关转录因子2（Runx2）、牙本质涎磷蛋白(DSPP)、牙本质基质蛋白1(DMP-1)以及Wnt1蛋白相对表达量。结果：与空白组、NC组比较，inhibitor组24 h、48 h、72 h吸光度值，GSK-3蛋白表达量降低，ALP活性值，矿化结节面积占比，-catenin、Runx2、DSPP、DMP-1以及Wnt1蛋白表达量升高（P0.05）；与inhibitor组比较，inhibitor+XAV939组24 h、48 h、72 h吸光度值，GSK-3蛋白表达量升高，ALP活性值，矿化结节面积占比，-catenin、Runx2、DSPP、DMP-1以及Wnt1蛋白表达量降低（P0.05）。结论：抑制miR-148a-3p表达可提升DPSCs增殖活力并促进其成骨分化，推测其作用机制与负向调控下游的靶基因Wnt1有关。关键词：微小RNA-148a-3p；无翅型MMTV整合位点家族蛋白1；-连环蛋白；牙髓干细胞；增殖；成骨分化中国图书分类号 R780.2 文献标识码 ADOI:10.19748/.kqxf.1009-3761.2023.5.003Effects of miR-148a-3p targeting Wnt1 gene on the proliferation and osteogenic differentiation of human dentalpulp stem cellsWU Dong1,ZHU Qing-qing1,GAO Zhen-jie2.(1.Oral Implant Department,Henan Provincial Stomatological Hospital,The First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China;2.Department of Oral and Maxillo-facial surgery,The First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)【Abstract】Objective:To observe the effect of microRNA(miRNA)-148a-3p on the proliferation and osteogenicdifferentiation of human dental pulp stem cells(DPSCs),and to explore the possible mechanism.Methods:Log phaseDPSCs were divided into theblank group,the NC group,the inhibitor group,and the inhibitor+XAV939 wingless MMTVintegration site family protein(Wnt)/-catenin(-catenin)signaling pathway inhibitor group.The blank group was nottreated,the NC group was transfected with negative control plasmid miR-148a-3p NC,the inhibitor group was transfectedwith miR-148a-3p inhibitor,and the inhibitor+XAV939 group was transfected with miR-148a-3p inhibitor and added withXAV939(40 mol/L).The expression of miR-148a-3p was detected by qRT-PCR 48 h after transfection.The proliferationactivity was detected by MTT method.Alkaline phosphatase(ALP)activity was analyzed by ELISA.Alizarin red stainingwas used to detect the mineralization capacity.Dual-luciferase assays were conducted to validate the miR-148a-3p targetinggenes.The relative expressions of glycogen synthase kinase 3(GSK-3),-catenin,Runt-related transcription factor 2(Runx2)and dentin sialophoshoprotein(DSPP),dentin matrix protein 1(DMP-1),Wnt1 were detected by western blotrespectively.Results:Compared with the blank group and NC group,the absorbance value at 24 h,48 h and 72 h,theexpression of GSK-3 protein were decreased,the activity value of ALP,the proportion of mineralized nodule area,andthe protein expression of-catenin and Runx2,DSPP,DMP-1,Wnt1 were increased in the inhibitor group(P0.05).Compared with the inhibitor group,the absorbance values at 24 h,48 h and 72 h,the expression of GSK-3 protein were吴东郑州大学第一附属医院河南省口腔医院口腔种植科主治医师郑州450052朱青青郑州大学第一附属医院河南省口腔医院口腔种植科主治医师郑州450052高振杰郑州大学第一附属医院口腔颌面外科副主任医师郑州450052335口腔颌面修复学杂志2023年9月第24卷第5期Chin J Prosthodont,September 2023,Vol.24,No.5increased,theALP activity value,the proportion of mineralized nodules,and the protein expressions of-catenin and Runx2,DSPP,DMP-1,Wnt1 were decreased in the inhibitor+XAV939 group(P0.05).Conclusion:Inhibiting the expression ofmiR-148a-3p can enhance the proliferation activity of DPSCs and promote their osteogenic differentiation.It is speculatedthat its mechanism is related to the negative regulation of the downstream target gene Wnt1.Key words:microRNA-148a-3p;wingless MMTV integration site family protein 1;-catenin;dental pulp stem cells;proliferation;osteogenic differentiation牙髓炎是临床最常见的口腔疾病之一，以自发性、阵发性剧烈疼痛为主要特征，分为可逆性及不可逆性1。目前临床治疗可逆性牙髓炎一般采用开髓引流术，后填充止痛药物，随之行盖髓术或牙髓切断术，以保牙髓消炎为目标；不可逆性牙髓炎则以根管治疗为主，可保存患牙并阻止病变扩散，但其亦造成敏感性丧失、脆性增加等问题2。近年来，以牙髓干细胞（dental pulp stem cells，DPSCs）移植为核心的牙组织再生工程技术崭露头角，其可在干预下进行成骨分化替代死亡的成牙本质细胞，恢复牙髓活力3。微小RNA（miRNA）是影响细胞生物学的重要调节因子，多项研究证实其在干细胞增殖及分化过程中扮演重要角色。有学者认为4，miR-148a-3p可通过调节骨髓间充质干细胞活力及成骨分化预防股骨头坏死，提示其具有成骨调节作用。然而目前关于其对DPSCs增殖活力及成骨分化的影响研究较少。本研究通过开展细胞实验，观察 miR-148a-3p 对 DPSCs 增殖活力及成骨分化的影响，并探讨可能机制。1.材料与方法1.1 细胞系 人DPSCs（上海酶联生物科技有限公司）。1.2 药物、主要试剂、仪器 miR-148a-3p NC质粒、miR-148a-3p inhibitor质粒、miR-148a-3pmimics质粒、LipofectamineTM3000试剂盒、双荧光素酶检测试剂盒（上海抚生实业有限公司），无翅型MMTV整合位点家族蛋白（wingless-type MMTV inte-gration site family members，Wnt）/-连环蛋白（-catenin）信号通路抑制剂XAV939（上海陶术生物科技有限公司），实时荧光定量聚合酶链反应试剂盒、MTT试剂盒、碱性磷酸酶（alkaline phosphatase，ALP）试剂盒（上海沪峥生物科技有限公司），兔抗人糖原合成酶激酶3（glycogen synthase kinase 3，GSK-3）、-catenin、Runt相关转录因子2（Runt-related transcription factor 2，Runx2）、牙本质涎磷蛋白(dentin sialophoshoprotein，DSPP)、牙本质基质蛋白1(dentin matrix protein 1，DMP-1)以及Wnt1抗体（日本MBL公司）。iMark酶标仪（美国Bio-rad公司），GENios酶标仪（瑞士Tecan公司），BX53显微镜（日本OLYMPUS公司）。1.3 细胞培养及鉴定 本研究已获得本单位伦理审查委员会批准，审批号为：KY-2023205。DPSCs常规培养，每隔两天弃掉旧培养基，加入新鲜含10%血清的培养基，显微镜下观察细胞，细胞密度达到80%以上时，进行细胞传代培养。取P3代DPSCs，胰酶消化，PBS重悬，取1106个细胞注入离心管内，低温离心3 min（1000 r/min，r=5 cm），吸弃上清并加入PBS，分别加入CD31、CD34、CD73、CD90抗体，遮光孵育20 min，经流式细胞仪分析四项表面标志物表达情况。1.4 qRT-PCR 法检测成骨诱导前后 miR-148a-3p表达量 取P3代DPSCs，PBS清洗，胰酶消化重悬，以1105个/mL接种至6孔板，加入完全培养液培养，细胞融合度达到90%左右，更换为成骨诱导培养液（10%胎牛血清的 DMEM 培养液+含 5mmol/L-甘油磷酸钠+50 mg/L维生素C+110-8mol/L地塞米松），2周后经qRT-PCR法检测诱导前后miR-148a-3p表达量。2-CT法分析计算miR-148a-3p相对表达量强度。实验所用引物：miR-148a-3p：Forward primer：5-ACCCGCTTCAAGGGAATTGG-3；Reverse primer：5-CTCTAGAGGCTGTGGTCGCC-3；U6：Forward primer：5-AATCGGGCCTGCTCAAGTG-3；Reverse primer：5-GAACAGAAAATGCGGACGGG-3。重复3次取均值。1.5 细胞转染、干预与分组 取 P3 代对数期DPSCs，PBS清洗，胰酶消化重悬，以1106个/mL接种至6孔板，加入完全培养液培养至贴壁，根据LipofectamineTM3000试剂盒说明书操作细胞转染，336Chin J Prosthodont,September 2023,Vol.24,No.5口腔颌面修复学杂志2023年9月第24卷第5期将miR-148a-3p抑制质粒miR-148a-3p inhibi-tor转染至 DPSCs，随机分为 inhibitor组、inhibi-tor+XAV939 组，将阴性对照质粒 miR-148a-3pNC转染至DPSCs，设为NC组，另取对数期DPSCs设为空白组。空白组、NC组、inhibitor组细胞置于DMEM培养基中常规培养，inhibitor+XAV939组培养基加入XAV939（40 mol/L）5，干预48 h后用于后续实验。1.6 qRT-PCR法检测细胞miR-148a-3p表达量 取各组细胞，操作同1.4。1.7 MTT法检测细胞增殖活力 取干预后各组细胞，以1106个/mL接种至6孔板，将培养板置于培养箱内，分别经过24、48、72 h后，取出培养板，每孔滴加MTT溶液20 L，经过4 h后滴加二甲基亚砜振荡溶解，测定吸光度（A）值，重复3次取均值。1.8 ELISA法检测细胞ALP活性 取干预后各组细胞，以1105个/mL接种至6孔板，加入成骨诱导培养液，2周后，PBS清洗，加入预冷RIPA裂解液，移至离心管内，离心，取上清液，即为待测样品，根据试剂盒说明书要求操作实验过程，依次抗体工作液及显色液，常温孵育20 min，加入硫酸溶液终止反应，于510 nm位置用酶标仪检测吸光度值，以各孔吸光度值与对照孔吸光度值之比，代表ALP活性值1.9 茜素红染色法检测细胞矿化能力 取干预后各组细胞，以1105个/mL接种至6孔板，加入成骨诱导培养液，2周后，胰蛋白酶消化细胞，3000 r/min离心10 min，PBS清洗，预冷75%酒精固定30 min，蒸馏水冲洗后浸入茜素红染液，30 min后PBS清洗，置于光镜下观察细胞矿化情况，Image Pro软件分析矿化结节面积占比。1.10 双荧光素酶实验验证miR-148a-3p靶向基因 经数据库TargetScan预测，miR-148a-3p与Wnt1 3 UTR存在结合位点，构建合成野生型Wnt13-UTR-WT及突变型Wnt1 3-UTR-MUT载体，与miR-148a-3p质粒（miR-148a-3p mimics、miR-148a-3p NC）共同转染至293T细胞，48 h后收集细胞，经试剂盒测定各细胞荧光酶相对活性。1.11 Western blot法检测细胞GSK-3、-catenin、Runx2、DSPP、DMP-1以及Wnt1蛋白相对表达量 取经成骨诱导2周后的各组细胞，加入预冷 RIPA 裂解液，移至离心管内，低温离心 20min（10000 r/min，r=10 cm），BCA 法测得裂解物蛋白浓度，沸水浴加热变性蛋白，SDS-PAGE将蛋白进行分离，之后将凝胶上的蛋白湿转至PVDF膜上，室温封闭，将目的蛋白抗体按照1：500的比例稀释后孵育蛋白条带，4孵育过夜，洗膜，二抗（1:2000）室温孵育，洗膜，ECL法进行蛋白条带显影，以GAPDH为内参，经成像分析仪扫描并分析GSK-3、-catenin、Runx2相对蛋白表达量。1.12 统计学处理 所有实验数据均用均数标准差(x s)表示，SPSS22.0软件用于数据分析和处理。所有数据均符合正态分布，且方差齐性，采用单因素方差分析，进一步两两比较时采用LSD-t检验，PC and c.505GT mutations on osteoblast dif-ferentiation via the WNT1/-catenin signaling path-wayJ.J Orthop Surg Res,2021,16(1):35916Qiu ZC,Zhang Y,Xiao HH,et al.8-prenylgenisteinexerts osteogenic effects via ER and Wnt-dependentsignaling pathwayJ.Exp Cell Res,2020,395(1):11218617Li XH,Chen FL,Shen HL.Salidroside promoted os-teogenic differentiation of adipose-derived stromal cellsthrough Wnt/-catenin signaling pathwayJ.J Or-thop Surg 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