ZEB1-AS1靶向微RNA-297调控脂多糖诱导的人牙周膜细胞损伤  

ZEB1-AS1 regulates lipopolysaccharide-induced injury of human periodontal ligament cells by targeting miR-297

作　　者：邢立婷 冯云霞[2] 马宇锋[1] 张亮[1] 马小杰 栗亚楠 王瑜[1] 刘名燕 Xing Liting;Feng Yunxia;Ma Yufeng;Zhang Liang;Ma Xiaojie;Li Yanan;Wang Yu;Liu Mingyan(Department of Stomatology,Second Hospital of Shanxi Medical University,Taiyuan,Shanxi 030001,China;Department of Orthodontics,Hospital of Stomatology,Shanxi Medical University,Taiyuan,Shanxi 030001,China;Department of Orthodontics,Suzhou Dental Doctor,Suzhou,Jiangsu 215021,China)

机构地区：[1]山西医科大学第二医院口腔科,太原030001 [2]山西医科大学口腔医院口腔正畸科,太原030001 [3]苏州牙博士口腔正畸科,江苏苏州215021

出　　处：《中华生物医学工程杂志》2021年第4期355-362,共8页Chinese Journal of Biomedical Engineering

基　　金：国家自然科学基金(81400566)。

摘　　要：目的探讨ZEB1-AS1对脂多糖(LPS)诱导的人牙周膜细胞损伤的影响和可能机制。方法体外培养人牙周膜细胞,分为对照组(Con组)、LPS组、LPS+si-NC组、LPS+si-ZEB1-AS1组、LPS+miR-NC组、LPS+miR-297组、LPS+si-ZEB1-AS1+anti-miR-NC组和LPS+si-ZEB1-AS1+anti-miR-297组,RT-PCR法检测细胞中ZEB1-AS1和miR-297表达水平,酶联免疫吸附法检测TNF-α和IL-1β表达水平,流式细胞术检测细胞凋亡,Western Blot检测细胞中Bcl-2和Bax蛋白表达水平。双荧光素酶报告基因实验验证ZEB1-AS1和miR-297调控关系。结果与Con组比较,LPS组ZEB1-AS1(3.65±0.24比1.00±0.07)、TNF-α[(8.87±0.73)ng/ml比(3.26±0.31)ng/ml]和IL-1β[(6.06±0.47)ng/ml比(1.96±0.14)ng/ml]水平、细胞凋亡率[(23.58±2.04)%比(7.69±0.45)%]和Bax蛋白水平(0.78±0.05比0.30±0.02)均升高(P<0.05),miR-297(0.49±0.05比1.00±0.05)和Bcl-2蛋白水平(0.21±0.02比0.63±0.04)均降低(P<0.05)。与LPS+si-NC组比较,LPS+si-ZEB1-AS1组TNF-α[(5.05±0.41)ng/ml比(8.95±0.58)ng/ml]和IL-1β[(3.35±0.27)ng/ml比(6.11±0.57)ng/ml]水平、细胞凋亡率[(11.53±1.07)%比(24.08±2.33)%]和Bax蛋白水平(0.39±0.04比0.79±0.07)降低(P<0.05),Bcl-2蛋白水平升高(0.56±0.04比0.20±0.02,P<0.05)。与LPS+miR-NC组比较,LPS+miR-297组TNF-α[(6.11±0.56)ng/ml比(9.14±0.62)ng/ml]和IL-1β[(3.93±0.27)ng/ml比(6.53±0.32)ng/ml]水平、细胞凋亡率[(14.04±1.05)%比(25.45±2.34)%]和Bax蛋白水平(0.43±0.03比0.80±0.05)降低(P<0.05),Bcl-2蛋白水平升高(0.52±0.05比0.19±0.02,P<0.05)。ZEB1-AS1在人牙周膜细胞中负调控miR-297表达。与LPS+si-ZEB1-AS1+anti-miR-NC组比较,LPS+si-ZEB1-AS1+anti-miR-297组TNF-α[(7.56±0.54)ng/ml比(4.94±0.35)ng/ml]和IL-1β[(5.25±0.32)ng/ml比(3.04±0.23)ng/ml]水平、细胞凋亡率[(19.92±1.05)%比(10.64±1.02)%]和Bax蛋白水平(0.67±0.04比0.39±0.03)升高(P<0.05),Bcl-2蛋白水平降低(0.30±0.02比0.58±0.04,P<0.05)。结论下调ZEB1-AS1可能通过靶向负调控miR-297降低LPS诱导的人牙周膜细胞炎症Objective To investigate the effect of ZEB1-AS1 on lipopolysaccharide(LPS)-induced injury of human periodontal ligament cells and its possible mechanism.Methods Human periodontal ligament cells were cultured in vitro and divided into control group(Con group),LPS group,LPS+si-NC group,LPS+si-ZEB1-AS1 group,LPS+miR-NC group,LPS+miR-297 group,LPS+si-ZEB1-AS1+anti-miR-NC group and LPS+si-ZEB1-AS1+anti-miR-297 group.RT-PCR was used to detect the ZEB1-AS1 and miR-297 expression in the cells;enzyme-linked immunosorbent assay was used to detect the expression of TNF-αand IL-1β;flow cytometry was used to detect cell apoptosis;Western Blot was used to detect the expression of Bcl-2 and Bax proteins in the cells.Dual luciferase reporter assay was used to verify the regulatory relationship between ZEB1-AS1 and miR-297.Results Compared with the Con group,the LPS group showed increases in expression levels of ZEB1-AS1(3.65±0.24 vs.1.00±0.07),TNF-α[(8.87±0.73)ng/ml vs.(3.26±0.31)ng/ml]and IL-1β[(6.06±0.47)ng/ml vs.(1.96±0.14)ng/ml],rate of apoptosis[(23.58±2.04)%vs.(7.69±0.45)%]and protein expression of Bax(0.78±0.05 vs.0.30±0.02)(P<0.05),but decreases in expression of miR-297(0.49±0.05 vs.1.00±0.05)and Bcl-2 protein(0.21±0.02 vs.0.63±0.04)(P<0.05).Compared with the LPS+si-NC group,the LPS+si-ZEB1-AS1 group showed increases in expression levels of TNF-α[(5.05±0.41)ng/ml vs.(8.95±0.58)ng/ml]and IL-1β[(3.35±0.27)ng/ml vs.(6.11±0.57)ng/ml],rate of apoptosis[(11.53±1.07)%vs.(24.08±2.33)%]and Bax protein(0.39±0.04 vs.0.79±0.07)(P<0.05),but increase in Bcl-2 protein expression(0.56±0.04 vs.0.20±0.02)(P<0.05).Compared with the LPS+miR-NC group,the LPS+miR-297 group showed decreases in expression levels of TNF-α[(6.11±0.56)ng/ml vs.(9.14±0.62)ng/ml]and IL-1β[(3.93±0.27)ng/ml vs.(6.53±0.32)ng/ml],rate of apoptosis[(14.04±1.05)%vs.(25.45±2.34)%]and Bax protein expression(0.43±0.03 vs.0.80±0.05)(P<0.05),but increase in Bcl-2 protein expression(0.52±0.05 vs.0.19±0.02)(P<0.05).ZEB1-AS1 negatively re

关 键 词：人牙周膜细胞 ZEB1-AS1 miR-297 炎症 细胞凋亡 

分 类 号：R781.42[医药卫生—口腔医学]