LINC00052靶向miR-145发挥对TNF-α诱导人关节软骨细胞损伤的保护作用  

LINC00052 Plays a Protective Role Against the Injury of TNF-αInduced Human Articular Chondrocytes by Targeting miR-145

作　　者：阿布都艾尼·热吾提 周文正[1] 车立新[1] 徐江波[1] 孙俊刚[1] Abuduaini·Rewuti;Zhou Wenzheng;Che Lixin(Department of Orthopedic Trauma,People's Hospital of Xinjiang Uygur Autonomous Region,Urumqi 830001,China)

机构地区：[1]新疆维吾尔自治区人民医院骨科中心创伤病区,新疆乌鲁木齐830001

出　　处：《实用骨科杂志》2021年第9期804-810,共7页Journal of Practical Orthopaedics

基　　金：新疆维吾尔自治区自然科学基金(2019D01C106)。

摘　　要：目的明确长链非编码RNA(long non-coding RNA,lncRNA)LINC00052在肿瘤坏死因子α(tumor necrosis factorα,TNF-α)诱导人关节软骨细胞损伤中的作用,并阐明其对miR-145的调控机制。方法利用0、10及30 ng/mL TNF-α诱导建立人C-20/A4关节软骨细胞损伤模型。采用实时荧光定量聚合酶链式反应(real time quantitative polymerase chain reaction,RT-qPCR)检测LINC00052与miR-145的表达及二者的相关性。通过特异性短发夹RNA干扰C-20/A4细胞中LINC00052的表达,采用四唑盐比色法[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,MTT]检测细胞增殖能力。采用蛋白质印迹检测半胱氨酸天冬氨酸蛋白酶3(caspase 3,CASP3)、cleaved CASP3、Bcl-2相关X(Bcl-2 associated X,BAX)蛋白的表达水平。采用酶联免疫吸附剂测定(enzyme linked immunosorbent assay,ELISA)检测炎症因子白介素(interleukin,IL)-1β、IL-6及IL-13的浓度。采用双荧光素酶报告基因实验分析LINC00052对miR-145的直接调控作用。采用拯救实验验证LINC00052靶向miR-145在TNF-α诱导C-20/A4细胞损伤中的作用。采用t检验与方差分析进行统计比较。结果LINC00052在0、10及30 ng/mL TNF-α诱导C-20/A4细胞中的表达水平呈上调趋势,而miR-145呈下调趋势,二者表达水平呈负相关(P<0.05)。干扰LINC00052降低TNF-α诱导C-20/A4细胞增殖能力,增加CASP3、cleaved CASP3及BAX的蛋白表达水平,并提高IL-1β、IL-6及IL-13的浓度(P<0.05)。miR-145是LINC00052的直接靶基因,干扰LINC00052上调C-20/A4细胞中miR-145的表达水平(P<0.05)。干扰miR-145部分逆转LINC00052抑制对TNF-α诱导C-20/A4细胞增殖、凋亡及炎症反应的影响(P<0.05)。结论LINC00052靶向miR-145发挥对TNF-α诱导人关节软骨细胞损伤的保护作用,其机制可能与促进增殖并抑制凋亡与炎症反应有关。Objective To identify the role of long non-coding RNA LINC00052 in the injury of tumor necrosis factor alpha(TNF-α)-induced human articular chondrocyte,and to elucidate its regulatory mechanism on miR-145.Methods The injury model of human C-20/A4 articular chondrocyte was induced and constructed by using 0,10 and 30 ng/mL of TNF-α.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression levels of LINC00052 and miR-145 and their expression correlation.The specific short hairpin RNA was used to interfere with the expression of LINC00052 in C-20/A4 cells,and cell proliferation ability was detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT).The protein expression levels of caspase-3(CASP3),cleaved CASP3,and Bcl-2 related X(BAX)were detected by Western blotting.The concentrations of inflammatory factors including interleukin(IL)-1β,-6 and-13 were detected by enzyme linked immunosorbent assay(ELISA).The direct regulation effect of LINC00052 on miR-145 was analyzed by dual luciferase reporter gene experiment.Rescue experiment was used to verify the role of LINC00052 by targeting miR-145 in the injury of TNF-α-induced C-20/A4 cells.Statistical analysis was compared by the t and analysis of variance.Results The expression level of LINC00052 in 0,10 and 30 ng/mL of TNF-α-induced C-20/A4 cells showed an upward trend,while miR-145 showed a downward trend,and their expression was negatively correlated(P<0.05).Interfering with LINC00052 reduced the proliferation of TNF-α-induced C-20/A4 cells,increased the protein expression levels of CASP3,cleared CASP3 and BAX,and elevated the concentrations of IL-1β,IL-6 and IL-13(P<0.05).miR-145 is a direct target gene of LINC00052,and interfering with LINC00052 upregulated the expression level of miR-145 in C-20/A4 cells(P<0.05).Interfering with miR-145 partially reversed the effect of LINC00052 suppression on the proliferation,apoptosis and inflammation in TNF-α-induced C-20/A4 cells(P<0.05).Conclusion LINC00052 plays a

关 键 词：LINC00052 软骨细胞 损伤 MIR-145 调控 

分 类 号：R684.3[医药卫生—骨科学]