微小RNA-455靶向调控泛素连接酶Cullin3活化核转录因子-κB在炎性神经痛中的作用及其机制  

The role and mechanism of microRNA-455 targeting Cullin3 to activate nuclear factor-κB in inflammatory neuralgia

作　　者：赵伟[1] 周思怡 徐大伟[2] Zhao Wei;Zhou Siyi;Xu Dawei(Department of Neurosurgery,Second Affiliated Hospital of Nantong University,Nantong 226001,China;Department of Orthopaedics,Second Affiliated Hospital of Nantong University,Nantong 226001,China)

机构地区：[1]南通大学第二附属医院神经外科,南通226001 [2]南通大学第二附属医院骨科,南通226001

出　　处：《中华实验外科杂志》2023年第9期1804-1807,共4页Chinese Journal of Experimental Surgery

基　　金：江苏省"333工程"项目(BRA2020204);江苏省六大人才高峰高层次人才(WSW-277)。

摘　　要：目的探讨微小RNA(miRNA,miR)-455靶向调控泛素连接酶Cullin3(CUL3)活化核转录因子-κB(NF-κB)在炎性神经痛中的作用及其机制。方法培养施万细胞(SCs),过表达miR-455、CUL3和反义miR-455以及慢病毒CUL3-短发卡RNA(shRNA)质粒沉默CUL3表达。运用实时荧光定量聚合酶链反应(qRT-PCR)检测miR-455、CUL3和NF-κB的RNA水平表达变化,运用酶联免疫吸附试验(ELISA)分析CUL3、NF-κB、肿瘤坏死因子(TNF)-α和白细胞介素(IL)-1β蛋白水平表达变化。组间比较采用t检验,多组比较采用单因素方差分析。结果过表达miR-455组CUL3蛋白表达低于对照组(6.25±1.54比32.56±5.18,F=59.694,P<0.01),而NF-κB(62.75±8.69比12.75±1.59,F=57.505,P<0.01)、TNF-α(186.55±17.84比52.14±6.23,F=66.576,P<0.01)和IL-1β(16.46±4.76比4.86±1.28,F=10.799,P<0.05)蛋白表达高于对照组。反义miR-455组CUL3蛋白表达高于反义miRC组(87.49±8.26比28.16±5.12,t=32.598,P<0.01),而反义miR-455组NF-κB(6.63±0.75比19.22±2.84,t=8.929,P<0.05)、TNF-α(26.23±2.14比63.17±7.76,t=7.739,P<0.05)和IL-1β(2.77±0.56比7.15±1.03,t=11.926,P<0.01)蛋白表达低于反义miRC组。同时,shCUL3组NF-κB(65.28±7.71比12.75±1.59,F=57.505,P<0.01)、TNF-α(172.95±19.57比52.14±6.23,F=66.576,P<0.01)和IL-1β(15.33±3.14比4.86±1.28,F=10.799,P<0.05)的蛋白表达高于对照组。而CUL3过表达组NF-κB(5.27±0.83比16.21±1.58,t=11.937,P<0.01)、TNF-α(19.72±2.04比55.04±4.38,t=14.4,P<0.01)和IL-1β(2.25±0.49比6.79±0.81,t=13.065,P<0.01)蛋白表达低于空转对照组。结论miR-455靶向调控CUL3从而促进了NF-κB蛋白的表达,在炎症性神经痛发病过程中发挥重要作用。Objective To investigate the role and mechanism of microRNA(miRNA,miR)-455 targeting Cullin 3(CUL3)-activated nuclear factor-κB(NF-κB)in inflammatory neuralgia.Methods Schwann cells(SCs)were cultured to overexpress miR-455,CUL3 and antisense miR-455,and the lentiviral CUL3-short hairpin RNA(shRNA)plasmid was used to silence CUL3 expression.The RNA levels of miR-455,CUL3 and NF-κB were detected by qRT-PCR,and the protein levels of CUL3,NF-κB,tumor necrosis factor(TNF)-αand interleukin(IL)-1βwere analyzed by enzyme linked immunosorbent assay(ELISA).Results Compared with the control group,down-regulation of CUL3(6.25±1.54 vs.32.56±5.18,F=59.694,P<0.01)by miR-455 resulted in the increased protein expression of NF-κB(62.75±8.69 vs.12.75±1.59,F=57.505,P<0.01),TNF-α(186.55±17.84 vs.52.14±6.23,F=66.576,P<0.01)and IL-1β(16.46±4.76 vs.4.86±1.28,F=10.799,P<0.05),whereas up-regulation of CUL3(87.49±8.26 vs.28.16±5.12,t=32.598,P<0.01)by antisense miR-455 caused down-regulation of NF-κB(6.63±0.75 vs.19.22±2.84,t=8.929,P<0.05),TNF-α(26.23±2.14 vs.63.17±7.76,t=7.739,P<0.05)and IL-1β(2.77±0.56 vs.7.15±1.03,t=11.926,P<0.01)proteins.Meanwhile,CUL3 silencing could also promote the protein expression of NF-κB(65.28±7.71 vs.12.75±1.59,F=57.505,P<0.01),TNF-α(172.95±19.57 vs.52.14±6.23,F=66.576,P<0.01)and IL-1β(15.33±3.14 vs.4.86±1.28,F=10.799,P<0.05),while overexpression of exogenous CUL3 could induce down-regulation of NF-κB(5.27±0.83 vs.16.21±1.58,t=11.937,P<0.01),TNF-α(19.72±2.04 vs.55.04±4.38,t=14.4,P<0.01)and IL-1β(2.25±0.49 vs.6.79±0.81,t=13.065,P<0.01)proteins.Conclusion MiR-455 targets and regulates CUL3 to promote the expression of NF-κB protein,which plays an important role in the pathogenesis of inflammatory neuralgia.

关 键 词：神经痛 施万细胞 微小RNA 泛素连接酶Cullin 3 核转录因子-ΚB 

分 类 号：R741[医药卫生—神经病学与精神病学]