长链非编码RNA LINC01133通过调控线粒体功能影响人牙周膜干细胞成牙骨质分化潜能的研究  

Long non-coding RNA LINC01133 regulates cementogenic differentiation of human periodontal ligament stem cells by modulating mitochondrial functions

作　　者：邓道坤 李璇 贺小涛[1] 孙海花[2] 田蓓敏[1] 陈发明[1] Deng Daokun;Li Xuan;He Xiaotao;Sun Haihua;Tian Beimin;Chen Faming(Department of Periodontology,School of Stomatology,The Fourth Military Medical University&State Key Laboratory of Military Stomatology&National Clinical Research Center for Oral Diseases&Shaanxi Engineering Research Center for Dental Materials and Advanced Manufacture,Xi′an 710032,China;Department of General Dentistry&Emergency,School of Stomatology,The Fourth Military Medical University&State Key Laboratory of Military Stomatology&National Clinical Research Center for Oral Diseases&Shaanxi International Joint Research Center For Oral Diseases,Xi′an 710032,China)

机构地区：[1]第四军医大学口腔医学院牙周病科,军事口腔医学国家重点实验室,国家口腔疾病临床医学研究中心,陕西省口腔生物工程技术研究中心,西安710032 [2]第四军医大学口腔医学院急诊与综合临床科,军事口腔医学国家重点实验室,国家口腔疾病临床医学研究中心,陕西省口腔疾病国际联合研究中心,西安710032

出　　处：《中华口腔医学杂志》2022年第12期1209-1216,共8页Chinese Journal of Stomatology

基　　金：国家自然科学基金(81970947, 82001102, 82170926, 82170958)。

摘　　要：目的探索长链非编码RNA(long noncoding RNA, lncRNA)LINC01133对人牙周膜干细胞(human periodontal ligament stem cells, hPDLSC)成牙骨质分化潜能的影响及其作用机制。方法收集2021年9月至2022年1月第四军医大学口腔医学院口腔颌面外科17~30岁10例就诊患者因正畸需要或因阻生拔除的牙齿共12颗, 从离体牙上提取hPDLSC, 分别转染靶向LINC01133小干扰RNA(small interfering RNA-LINC01133, si-LINC01133)或阴性对照小干扰RNA(small interfering RNA-negative control, si-NC), 以转染si-LINC01133为实验组, 转染si-NC为阴性对照组。利用实时荧光定量PCR(real-time quantitative PCR, RT-qPCR)检测si-LINC01133的沉默效率;通过蛋白质印迹法检测hPDLSC成牙骨质分化相关蛋白包括骨涎蛋白(bone sialoprotein, BSP)、牙骨质附着蛋白(cementum attachment protein, CAP)、牙骨质蛋白1(cementum protein-1, CEMP-1)的表达;利用流式细胞术和线粒体超氧化物指示剂MitoSOX检测细胞线粒体活性氧产量;通过JC-1荧光染色法检测线粒体膜电位水平;利用蛋白质印迹法检测线粒体呼吸链复合体蛋白包括NADH脱氢酶[泛醌]1β亚单位8(NADH dehydrogenase[ubiquinone]1 beta subcomplex subunit 8, NDUFB8)、琥珀酸脱氢酶亚单位A(succinate dehydrogenase complex flavoprotein subunit A, SDHA)、泛醌-细胞色素c还原酶核心蛋白1(ubiquinol-cytochrome c reductase core protein 1, UQCR1)、细胞色素c氧化酶亚单位4亚型1(cytochrome c oxidase subunit 4 isoform 1, COXⅣ)、ATP合成酶F1亚单位α(ATP synthase F1 subunit alpha, ATP5A)的表达水平。结果 hPDLSC的LINC01133表达水平被si-LINC01133有效沉默(阴性对照组:1.000±0.000, 实验组:0.385±0.128)(t=10.72, P<0.01), 沉默效率超过60%。LINC01133沉默后, hPDLSC BSP表达水平显著下降(阴性对照组:1.000±0.000, 实验组:0.664±0.179)(t=4.62, P<0.01);CAP表达水平显著下降(阴性对照组:1.000±0.000, 实验组:0.736±0.229)(t=2.83, P<0.05)。LINC01133沉默后, hPDLSC线粒Objective To investigate the effects of long non-coding RNA(lncRNA)LINC01133 on the cementogenic differentiation of human periodontal ligament stem cells(hPDLSC)and the underlying mechanism.Methods A total of 12 teeth were harvested from 10 patients aged 17-30 years in the Department of Oral and Maxillofacial Surgery,School of Stomatology,The Fourth Military Medical University for impacted or orthodontic reasons from September 2021 to January 2022.The hPDLSCs were isolated from the teeth and transfected with small interfering RNA-LINC01133(si-LINC01133)or small interfering RNA-negative control(si-NC).The si-LINC01133 was regarded as the experimental group,and the si-NC was regarded as the control one.The silencing efficiency of LINC01133 in the hPDLSCs was evaluated by real-time quantitative PCR(RT-qPCR).Western blotting was used to detect the protein expression levels of cementogenic differentiation-related factors including bone sialoprotein(BSP),cementum attachment protein(CAP),and cementum protein-1(CEMP-1).Mitochondrial reactive oxygen species(mtROS)production was assessed using the MitoSox by flow cytometry.Mitochondrial membrane potential(MMP)was detected by JC-1 fluorescence staining.Mitochondrial respiratory chain complexes proteins including NADH dehydrogenase[ubiquinone]1 beta subcomplex subunit 8(NDUFB8),succinate dehydrogenase complex flavoprotein subunit A(SDHA),ubiquinol-cytochrome c reductase core protein 1(UQCR1),cytochrome c oxidase subunit 4 isoform 1(COXⅣ),and ATP synthase F1 subunit alpha(ATP5A)were evaluated by Western blotting.Results The expression levels of LINC01133 could be suppressed by more than 60%with si-LINC01133(control group:1.000±0.000,experimental group:0.385±0.128)(t=10.72,P<0.01).Suppression of LINC01133 in hPDLSCs decreased the levels of cementogenic differentiation-related proteins including BSP(control group:1.000±0.000,experimental group:0.664±0.179)(t=4.62,P<0.01)and CAP(control group:1.000±0.000,experimental group:0.736±0.229)(t=2.83,P<0.05).Suppression of LINC0

关 键 词：牙周炎 细胞分化 成牙骨质分化 长链非编码RNA 牙周膜干细胞 线粒体功能 

分 类 号：R781.42[医药卫生—口腔医学]