摘要
目的探讨微小RNA-320-3p(miR-320-3p)是否通过调控高迁移率族蛋白1的表达调控小鼠关节软骨细胞外基质降解过程,并探索其调控机制。方法2020年11月至2023年3月,获取6只5~7 d重约6 g的C57BL/6乳鼠关节软骨细胞,通过白细胞介素-1β(IL-1β)刺激软骨细胞并进行培养,运用TRIzol法提取软骨细胞总RNA;采用实时荧光定量反转录聚合酶链式反应(RT-PCR)检测miR-320-3p、高迁移率族蛋白B1(HMGB1)、基质金属蛋白酶(MMP)-13以及Col2a1的表达情况;分别转染miR-320-3P模拟物(mimic组)及空白对照(NC mimic组)、miR-320-3p抑制物(inhibitor组)及空白对照(NC inhibitor组),观察miR-320-3p调控软骨细胞HMGB1、MMP-13以及Col2a1的表达;蛋白质印迹法(Western blot)实验检测HMGB1的表达水平;双荧光素酶报告实验验证miR-320-3p与HMGB1 mRNA 3’非编码区(UTR)的结合情况,采用独立样本t检验进行分析。结果RT-PCR结果显示,mimic组miRNA-320-3p表达高于NC mimic组(67.49±2.97比1.03±0.19),差异有统计学意义(t=38.70,P<0.01);inhibitor组miRNA-320-3p表达低于NC inhibitor组(0.47±0.07比1.04±0.10),差异有统计学意义(t=8.26,P<0.01);经IL-1β处理的软骨细胞miRNA-320-3p表达量低于正常组织水平(0.51±0.04比1.01±0.22),差异有统计学意义(t=3.96,P<0.05);HMGB1的表达量高于正常组织水平(4.64±0.72比1.03±0.29),差异有统计学意义(t=8.12,P<0.01);Col2a1的表达低于正常组织水平(0.36±0.05比1.00±0.10),差异有统计学意义(t=10.32,P<0.01);MMP-13的表达高于正常组织水平(6.22±0.82比1.00±0.15),差异有统计学意义(t=10.86,P<0.01)。Western blot实验显示mimic组HMGB1的表达低于NC mimic组(0.48±0.02比0.85±0.05),差异有统计学意义(t=13.28,P<0.01),inhibitor组HMGB1的表达高于NC inhibitor组(0.90±0.05比0.56±0.06),差异有统计学意义(t=7.39,P<0.01)。转染miR-320-3p模拟物后,mimic组HMGB1的表达低于NC mimic组(0.43±0.07比1.02±0.25),差异有统计学意义(t=3.96,P<0.05);mimic组MMP-13的表达低于NC mimic组(0.65±0.11比1.07±0.16),差异有统计学意义(t=3.76,P<0.05);mimic组Col2a1的表达高于NC mimic组(2.10±0.29比1.07±0.20),差异有统计学意义(t=5.09,P<0.01);而转染miRNA-320-3p抑制物后,inhibitor组HMGB1的表达高于NC inhibitor组(2.89±0.23比1.01±0.20),差异有统计学意义(t=10.58,P<0.01);inhibitor组MMP-13的表达高于NC inhibitor组(2.25±0.31比1.02±0.20),差异有统计学意义(t=5.78,P<0.01);inhibitor组Col2a1的表达低于NC inhibitor组(0.38±0.06比1.02±0.17),差异有统计学意义(t=6.03,P<0.01)。过表达miRNA-320-3p后突变型的结合位点荧光素酶活性低于对照组,差异无统计学意义(2.91±0.91比3.19±1.00,t=0.36,P>0.05),而野生型的结合位点荧光素酶活性低于对照组(2.73±0.71比3.83±1.00),差异有统计学意义(t=1.55,P<0.01)。结论miR-320-3p可通过靶向软骨细胞HMGB1的表达,介导软骨细胞外基质的降解过程进而抑制或缓解骨关节炎的发生。
Objective Study whether microRNA-320-3p regulates the extracellular matrix degradation process of mouse articular cartilage cells by regulating the expression of high mobility group protein 1,and explore its regulatory mechanism.Methods From November 2020 to March 2023,obtain joint chondrocytes from 6 C57BL/6 neonatal mice aged 5-7 days and weighing approximately 6 g.Chondrocytes were stimulated with interleukin-1β(IL-1β)and cultured,and total RNA was extracted from chondrocytes using the Trizol method.Real-time fluorescence quantitative reverse transcription polymerase chain reaction(RT-PCR)was used to detect the expression of miRNA-320-3p,HMGB1,MMP13,and Col2a1.Transfect miRNA-320-3P mimic(mimic group)and blank control(NC mimic group),miRNA-320-3p inhibitor(inhibitor group)and blank control(NC inhibitor group)separately.Observing the regulation of HMGB1,MMP13,and Col2a1 expression in chondrocytes by miRNA-320-3p.Western blotting experiment was used to detect the expression level of HMGB1.The dual luciferase reporter assay was used to verify the binding of miR-320-3p to the 3’non coding region(UTR)of HMGB1 mRNA,and independent sample t-test was employed for analysis.Results The RT-PCR results showed that the expression of miRNA-320-3p in the mimic group was higher than that in the NC mimic group(67.49±2.97 vs.1.03±0.19),and the difference was statistically significant(t=38.70,P<0.01).The expression of miRNA-320-3p in the inhibitor group was lower than that in the NC inhibitor group(0.47±0.07 vs.1.04±0.10),and the difference was statistically significant(t=8.26,P<0.01).The expression level of miRNA-320-3p in chondrocytes treated with IL-1βwas lower than that in normal tissues(0.51±0.04 vs.1.01±0.22),and the difference was statistically significant(t=3.96,P<0.05).The expression level of HMGB1 was higher than that of normal tissues(4.64±0.72 vs.1.03±0.29),and the difference was statistically significant(t=8.12,P<0.01).The expression of Col2a1 was lower than the normal tissue level(0.36±0.05 vs.1.00±0.10),and the difference was statistically significant(t=10.32,P<0.01).The expression of MMP-13 was higher than the normal tissue level(6.22±0.82 vs.1.00±0.15),and the difference was statistically significant(t=10.86,P<0.001).Western blotting experiments showed that the expression of HMGB1 in the mimic group was lower than that in the NC mimic group(0.48±0.02 vs.0.85±0.05),and the difference was statistically significant(t=13.28,P<0.01).The expression of HMGB1 in the inhibitor group was higher than that in the NC inhibitor group(0.90±0.05 vs.0.56±0.06),and the difference was statistically significant(t=7.39,P<0.01).After transfection with miR-320-3p mimic,the expression of HMGB1 in the mimic group was lower than that in the NC mimic group(0.43±0.07 vs.1.02±0.25),and the difference was statistically significant(t=3.96,P<0.05).The expression of MMP-13 in the mimic group was lower than that in the NC mimic group(0.65±0.11 vs.1.07±0.16),and the difference was statistically significant(t=3.76,P<0.05).The expression of Col2a1 in the mimic group was higher than that in the NC mimic group(2.10±0.29 vs.1.07±0.20),and the difference was statistically significant(t=5.09,P<0.01).After transfection with miRNA-320-3p inhibitor,the expression of HMGB1 in the inhibitor group was higher than that in the NC inhibitor group(2.89±0.23 vs.1.01±0.20),and the difference was statistically significant(t=10.58,P<0.01).The expression of MMP-13 in the inhibitor group was higher than that in the NC inhibitor group(2.25±0.31 vs.1.02±0.20),and the difference was statistically significant(t=5.78,P<0.01).The expression of Col2a1 in the inhibitor group was lower than that in the NC inhibitor group(0.38±0.06 vs.1.02±0.17),and the difference was statistically significant(t=6.03,P<0.01).The luciferase activity at the binding site of miRNA-320-3p mutant after overexpression was lower than that of the control group,the difference was not statistically significant(2.91±0.91 vs.3.19±1.00),and(t=0.36,P>0.05).In contrast,the luciferase activity of the wild-type binding site was lower than that of the control group(2.73±0.71 vs.3.83±1.00),and the difference was statistically significant(t=1.55,P<0.01).Conclusion MiRNA-320-3p can inhibit or alleviate the occurrence of osteoarthritis by targeting the expression of HMGB1 in chondrocytes,mediating the degradation process of extracellular matrix in chondrocytes.
作者
蓝奉军
孙红
周江湖
徐昌超
吴鸿斌
杨华
Lan Fengjun;Sun Hong;Zhou Jianghu;Xu Changchao;Wu Hongbin;Yang Hua(Department of Orthopaedics,Affiliated Hospital of Guizhou Medical University,Guiyang 550004,China)
出处
《中华实验外科杂志》
2025年第7期1221-1225,共5页
Chinese Journal of Experimental Surgery
基金
贵州医科大学附属医院国家自然科学基金青年基金培育计划项目(gyfynsfc-2021-12)
贵州省科技厅基础研究计划(黔科合基础-ZK[2021]391)
贵州省卫生健康委科学技术基金(gzwkj2021-261)
贵州省研究生科研基金项目(YJSKYJJ[2021]157)贵州省科技厅基础研究计划(黔科合基础-ZK[2023]一般344)。
