摘要
目的研究miR-130a对白介素-1β(interleukin-1β,IL-1β)诱导骨关节炎细胞模型的保护作用及机制。方法培养大鼠骨关节软骨细胞ATDC5,用不同浓度IL-1β(0、1、5、10 ng/ml)刺激后检测miR-130a表达的变化;另外将ATDC5细胞分为对照组、miR-NC组、IL-1β+miR-NC组、IL-1β+miR-130a组、Ad-NC组、Ad-NC+IL-1β组、Ad-NC+IL-1β+miR-130a组、Ad-PTEN+IL-1β+miR-130a组,用10 ng/ml IL-1β活力并转染阴性对照(NC)miR、miR-130a、NC腺病毒、过表达PTEN的腺病毒,MTS法检测细胞活力A490,TUNEL法检测凋亡率,western blot检测PTEN、p-PI3K、p-AKT表达水平,双荧光素酶报告基因验证miR-130a靶向调控PTEN基因。结果1、5、10 ng/ml IL-1β刺激ATDC5后miR-130a的表达水平分别为(0.74±0.15)、(0.62±0.09)、(0.54±0.08),均低于0 ng/ml IL-1β组(0.94±0.14)(P<0.05)且10 ng/ml IL-1β刺激后miR-130a表达降低最显著。IL-1β+miR-NC组A490水平、p-PI3K及p-AKT表达水平分别为(0.45±0.09)、(0.40±0.09)、(0.60±0.10),低于对照组(0.92±0.21)、(0.89±0.16)、(0.97±0.17)以及miR-NC组(0.88±0.19)、(0.93±0.18)、(1.08±0.22),凋亡率、PTEN表达水平分别为(25.61±5.52)%、(0.95±0.18),高于对照组(3.84±0.95)%、(0.52±0.09)以及miR-NC组(4.41±0.84)%、(0.60±0.12)(P<0.05);IL-1β+miR-130a组A490水平、p-PI3K及p-AKT表达水平分别为(1.05±0.26)、(0.85±0.17)、(1.04±0.21),高于IL-1β+miR-NC组,凋亡率、PTEN表达水平分别为(8.95±1.48)%、(0.56±0.10),低于IL-1β+miR-NC组(P<0.05);miR-130a组野生型PTEN双荧光素酶报告基因的荧光活力为(0.38±0.09),低于miR-NC组(0.83±0.15)(P<0.05);Ad-PTEN+IL-1β+miR-130a组的A490水平为(0.39±0.08)、低于Ad-NC+IL-1β+miR-130a组(0.86±0.16),凋亡率为(23.11±6.95)%、高于Ad-NC+IL-1β+miR-130a组(6.58±1.21)%(P<0.05)。结论miR-130a对IL-1β诱导骨关节炎细胞模型具有保护作用,靶向调控PTEN表达并增强细胞活力、抑制细胞凋亡是可能的分子机制。
Objective To study protective effects and mechanism of miR-130a on interleukin-1β(IL-1β)induced osteoarthritis cell model.Methods Rat chondrocytes ATDC5 were cultured in vitro,and the expression of miR-130a was detected after stimulation with different concentrations of IL-1β(0,1,5,10 ng/ml).In addition,ATDC5 cells were divided into control group,miR-NC group,IL-1β+miR-NC group,IL-1β+miR-130a group,AD-NC group,AD-NC+IL-1βgroup,AD-NC+IL-1β+miR-130a group,Ad-PTEN+IL-1β+miR-130a group.All were treated with 10 ng/ml IL-1βand transfected with negative control(NC)miR,miR-130a,NC adenovirus,PTEN overexpressed adenovirus.Cell viability was detected by MTS,apoptosis rate was detected by TUNEL,expression levels of PTEN,p-PI3K,p-AKT were detected by western blot,and miR-130a targeted PTEN gene was verified by double luciferase reporter gene.Results After 1,5 and 10 mg/ml IL-1βstimulation,expression levels of miR-130a in ATDC5 were(0.74±0.15),(0.62±0.09),(0.54±0.08),which were lower than(0.94±0.14)of 0 ng/mlIL-1βgroup(P<0.05)while the expression of miR-130a decreased most significantly after 10 ng/ml IL-1βstimulation.The level of A490,the expression of p-PI3K and p-AKT in IL-1β+miR-NC group were(0.45±0.09),(0.40±0.09),(0.60±0.10),which were lower than(0.92±0.21),(0.89±0.16),(0.97±0.17)in control group and(0.88±0.19),(0.93±0.18),(1.08±0.22)in miR-NC group.The apoptosis rate and the expression of PTEN were(25.61±5.52)%,(0.95±0.18),which were higher than(3.84±0.95)%,(0.52±0.09)in control group and(4.41±0.84)%,(0.60±0.12)in miR-NC group.The level of A490,the expression of p-PI3K and p-AKT in IL-1β+miR-130a group were(1.05±0.26),(0.85±0.17),(1.04±0.21),which were higher than those in IL-1β+miR-NC group.The apoptosis rate and the expression of PTEN were(8.95±1.48)%,(0.56±0.10),which were lower than those in IL-1β+miR-NC group.The fluorescence activity of PTEN double luciferase reporter gene in miR-130a group was(0.38±0.09),which was lower than that of miR-NC group(0.83±0.15).The level of A
作者
卢伟广
汤善华
王灿斌
LU Wei-guang;TANG Shan-hua;WANG Can-bin(The Third Ward of Orthopedics,Logistic Support Forces of the Chinese People's Liberation Army 908 Hospital,Nanchang,Jiangxi,335000,China)
出处
《中国骨与关节杂志》
CAS
2021年第7期554-560,共7页
Chinese Journal of Bone and Joint
