miR-455-3p调控sirt1表达对大鼠椎间盘髓核细胞增殖及凋亡的影响  

Influence of miR-455-3p on proliferation and apoptosis of rat intervertebral disc nucleus pulposus cells by regulating sirt1 expression

作　　者：唐家国 李晨芳 左斌 何精选 夏晓枫 王鹏 邹凯 娄文杰 TANG Jia-guo;LI Chen-fang;ZUO Bin;HE Jing-xuan;XIA Xiao-feng;WANG Peng;ZOU Kai;LOU Wen-jie(Department of Orthopaedics,Changjiang Hospital of the Yangtze River Shipping·Wuhan Brain Hospital,Wuhan,Hubei 430000,China;Department of Geriatrics,Changjiang Hospital of the Yangtze River Shipping·Wuhan Brain Hospital,Wuhan,Hubei 430000,China)

机构地区：[1]长江航运总医院·武汉脑科医院骨外科,湖北武汉430000 [2]长江航运总医院·武汉脑科医院老年科,湖北武汉430000

出　　处：《颈腰痛杂志》2024年第1期1-8,13,共9页The Journal of Cervicodynia and Lumbodynia

基　　金：湖北省卫生健康科研基金资助项目(编号:WJ2019H388);武汉市卫生健康科研基金资助项目(编号:WX19Q41)。

摘　　要：目的探讨miR-455-3p调控沉默信息调节因子2同源蛋白1(silent mating type information regulation 2 homolog 1,sirt1)表达对大鼠椎间盘髓核(nucleus pulposus,NP)细胞增殖及凋亡的影响。方法采用Ⅱ型胶原酶从大鼠腰椎椎间盘中分离NP细胞,并用甲苯胺蓝和Ⅱ型胶原(CollagenⅡ)免疫荧光染色对其鉴定。NP细胞分成Ctrl组、白细胞介素-1β(interleukin-1β,IL-1β)组、IL-1β+NC inhibitor组、IL-1β+miR-455-3p inhibitor组、IL-1β+miR-455-3p inhibitor+si-NC组、IL-1β+miR-455-3p inhibitor+si-sirt1组,除Ctrl组外的五组细胞均使用10 ng/mL的IL-1β诱导NP细胞退变模型。实时荧光定量聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction,qRT-PCR)检测miR-455-3p及sirt1 mRNA水平;细胞计数试剂盒8(cell counting kit 8,CCK-8)检测增殖;JC-1染色检测线粒体膜电位;Hoechst染色检测凋亡;蛋白免疫印迹检测sirt1、增殖(PCNA、Ki67、Cyclin D1)和凋亡(Bcl-2、Bax、Cleaved-caspase 3)相关蛋白及CollagenⅡ、Aggrecan表达;双荧光素酶报告分析实验验证miR-455-3p与sirt1的靶向关系。结果大鼠椎间盘NP细胞被成功分离。与IL-1β组、IL-1β+NC inhibitor组比较,IL-1β+miR-455-3p inhibitor组miR-455-3p表达、凋亡率、Bax、Cleaved-caspase 3表达及Bax/Bcl-2比值下降,细胞活力、PCNA、Ki67、Cyclin D1、Bcl-2、CollagenⅡ、Aggrecan表达升高(P<0.05)。miR-455-3p直接靶向负调控sirt1表达。干扰sirt1可逆转抑制miR-455-3p表达对NP细胞增殖、凋亡的影响。结论抑制miR-455-3p表达可通过靶向调控sirt1,抑制IL-1β诱导的NP细胞凋亡并促进NP细胞增殖。Objective To investigate the influence of miR-455-3p on the proliferation and apoptosis of rat intervertebral disc nucleus pulposus(NP)cells by regulating the expression of silent information regulator 2 homolog 1(sirt1).Methods NP cells were isolated from rat lumbar intervertebral discs using collagenase type II and identified by immunofluorescence staining with toluidine blue and collagen type II(collagen II).NP cells were randomly grouped into Ctrl group,interleukin-1β(IL-1β)group,IL-1β+NC inhibitor group,IL-1β+miR-455-3p inhibitor group,IL-1β+miR-455-3p inhibitor+si-NC group,and IL-1β+miR-455-3p inhibitor+si-sirt1 group,then 10 ng/mL IL-1β was used to induce the NP cell degeneration model in the five groups except the Ctrl group.Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)was performed to measure miR-455-3p and sirt1 mRNA levels;cell counting kit 8(CCK-8)was used to detect proliferation;JC-1 staining was carried out to assess mitochondrial membrane potential;Hoechst staining was employed to detect the apoptosis;Western blot was performed to measure the expression of sirt1,proliferation(PCNA,Ki67,cyclin D1)and apoptosis(Bcl-2,Bax,cleaved caspase-3)related proteins and collagen II and aggrecan expression;dual-luciferase reporter assay experiment was applied to verify the targeting relationship between miR-455-3p and sirt1.Results Rat intervertebral disc NP cells were successfully isolated.Compared with the IL-1β group and IL-1β+NC inhibitor group,miR-455-3p expression,apoptosis rate,Bax,cleaved caspase-3 expression and Bax/Bcl-2 ratio decreased in the IL-1β+miR-455-3p inhibitor group,while cell viability,and the expression of PCNA,Ki67,cyclin D1,Bcl-2,collagen II and aggrecan increased(P<0.05).MiR-455-3p directly targeted and negatively regulated sirt1 expression.Interfering with sirt1 was able to reverse the effects of inhibiting the expression of miR-455-3p on the proliferation and apoptosis of NP cells.Conclusion Inhibition of miR-455-3p expression can inhibit IL-1β-induc

关 键 词：miR-455-3p 沉默信息调节因子2同源蛋白1 大鼠 椎间盘髓核细胞 增殖 凋亡 

分 类 号：R681.5[医药卫生—骨科学]