基于NF-κB信号通路探讨miR-155调控的靶基因CEBPβ对颈椎病大鼠椎间盘软骨细胞凋亡和炎症反应的影响OACSTPCD
Effect of miR-155 Target Gene CEBPβ on Apoptosis and Inflammato-ry Response of Intervertebral Disc Chondrocytes in Rat with Cervical Spondylosis through Regulation of NF-κB Signaling Pathway
目的 探讨微小RNA(miR)-155 的靶向CCAAT增强子结合蛋白β(C/EBPβ)对颈椎病大鼠椎间盘软骨细胞凋亡和炎症反应的作用和潜在机制.方法 将40 只成年雄性SD大鼠随机分为 4 组,分别为假手术组、颈椎病组、颈椎病+过表达C/EBPβ组、颈椎病+空载体组,每组10 只.将大鼠软骨细胞系atdc5 细胞分为6 组,分别为对照组、TNF-α组、空载体+TNF-α组、C/EBPβ过表达+TNF-α组、mimic-NC+C/EBPβ过表达+TNF-α组、mimic miR-155+C/EBPβ过表达+TNF-α组.通过qRT-PCR和蛋白质印迹实验检测各组大鼠软骨组织和 atdc5 细胞中 miR-155、凋亡相关蛋白(cleaved-caspase3、Bax、Bcl-2、PPARγ)、NF-κB信号通路蛋白(p-NF-κB P65、NF-κB P65、IκB)、促炎因子(TNF-α、IL-6、IL-1β)以及C/EBPβ的表达.用双荧光素酶报告基因实验检测miR-155 对C/EBPβ表达的靶向调控作用.结果 与假手术组比,颈椎病组软骨中miR-155、cleaved-caspase3、Bax、PPARγ、p-NF-κB P65、TNF-α、IL-6、IL-1β的表达均上调,而Bcl-2、IκB、C/EBPβ均下调(P<0.05).与颈椎病+空载体组比,颈椎病+过表达 C/EBPβ组软骨中cleaved-caspase3、Bax、PPARγ、p-NF-κB P65、TNF-α、IL-6、IL-1β的表达均下调,而Bcl-2、IκB、C/EBPβ均上调(P<0.05),但miR-155 变化不显著(P>0.05).颈椎病大鼠软骨中miR-155 与C/EBPβ的表达水平显著负相关(r=-0.721,P<0.05).与对照组比,TNF-α 组中 miR-155、cleaved-caspase3、Bax、PPARγ、p-NF-κB P65、IL-6、IL-1β的表达均上调(P<0.05),而Bcl-2、IκB、C/EBPβ均下调(P<0.05).与空载体+TNF-α组比,C/EBPβ过表达+TNF-α组中 cleaved-caspase3、Bax、PPARγ、p-NF-κB P65、IL-6、IL-1β的表达均下调,而Bcl-2、IκB、C/EBPβ均上调(P<0.05),但miR-155 变化不显著(P>0.05).与mimic-NC+C/EBPβ过表达+TNF-α组相比,mimic+C/EBPβ过表达+TNF-α组中miR-155、cleaved-caspase3、Bax、PPARγ、p-NF-κB P65、IL-6、IL-1β的表达均上调(P<0.05),而Bcl-2、IκB、C/EBPβ均下调(P<0.05).双荧光素酶报告基因实验显示软骨细胞中miR-155 的靶基因是CEBPβ.结论 miR-155 通过靶向抑制C/EBPβ促进颈椎病大鼠椎间盘软骨组织和软骨细胞中NF-κB信号通路的激活,并加快细胞的凋亡和炎症反应.
Objective To explore the role and potential mechanisms of CCAAT-enhancer-binding protein β(C/EBPβtargeted by microRNA(miR)-155 in apoptosis and inflammatory re-sponse of rat intervertebral disc chondrocytes with cervical spondylosis.Methods Forty adult male SD rats were randomly divided into 4 groups with 10 rats in each group:Sham group,Cervical Spondylosis group,Cervical Spondylosis+C/EBPβ overexpression group,and Cervical Spondylosis+empty vector group.Rat chondrocyte cell line,atdc5 cells,were divided into 6 groups:control group,tumor necrosis factor α(TNF-α)stimulation group,empty vector+TNF-α group,C/EBPβ overexpression+TNF-α group,mimic-NC+C/EBPβ overexpression+TNF-α group,and miR-155 mimic+C/EBPβ overexpression+TNF-α group.The expression of miR-155,apoptosis-re-lated proteins(cleaved-caspase3,Bax,Bcl-2,PPARγ),NF-κB signaling pathway proteins(p-NF-κB P65,NF-κB P65,IκB),pro-inflammatory cytokines(TNF-α,IL-6,IL-1β),and C/EBPβ in rat cartilage tissues and atdc5 cells were detected using qRT-PCR and Western blotting ex-periments.Dual-luciferase reporter gene assay was conducted to determine the targeted regulatory effect of miR-155 on C/EBPβ expression.Results miR-155,cleaved-caspase3,Bax,PPARγ,p-NF-κB P65,TNF-α,IL-6,and IL-1β were upregulated in the Cervical Spondylosis group when compared with those in the Sham group,while Bcl-2,IκB,and C/EBPβ were downregulated in the Cervical Spondylosis group(P<0.05)Ceaved-caspase3,Bax,PPARγ,p-NF-κB P65,TNF-α,IL-6,and IL-1β were downregulated in the Cervical Spondylosis+C/EBPβ overexpression group when compared with those in the Cervical Spondylosis+empty vector group,while Bcl-2,IκB,and C/EBPβ were upregulated in the Cervical Spondylosis+C/EBPβ overexpression group(P<0.05),but there were no significant change in miR-155 expression level(P>0.05).There was a significant negative correlation between the expression levels of miR-155 and C/EBPβ in the cartilage of rats with cervical spondylosis(r=-0.721,P<0.05).The TNF-α group showed up-regulations of miR-155,cleaved-caspase3,Bax,PPARγ,p-NF-κB P65,IL-6,and IL-1β ex-pression(P<0.05),and downregulations of Bcl-2,IκB,and C/EBPβ expression when com-pared with the control group(P<0.05).The C/EBPβ overexpression+TNF-α group showed down-regulations of cleaved-caspase3,Bax,PPARγ,p-NF-κB P65,IL-6,and IL-1β expression,and upregulations of Bcl-2,IκB,and C/EBPβ expression when compared with the empty vector+TNF-α group(P<0.05),but no significant change was observed in miR-155 expression level(P>0.05).The mimic+C/EBPβ overexpression+TNF-α group showed upregulations of miR-155,cleaved-caspase3,Bax,PPARγ,p-NF-κB P65,IL-6,and IL-1β expression(P<0.05),and downregulations of Bcl-2,IκB,and C/EBPβ expression when compared with the mimic-NC+C/EBPβ overexpression+TNF-α group(P<0.05).Dual-luciferase reporter gene assay confirmed CEBPβ as a target gene of miR-155 in chondrocytes.Conclusion miR-155 promotes the activation of the NF-κB signaling pathway in intervertebral disc cartilage tissues and chondrocytes in rat with cervical spondylosis by inhibiting the target gene CEBPβ,which leads to enhanced apoptosis and in-flammatory response.
纳青;吐尔逊娜依·阿布都热依木;吴刚
乌鲁木齐市友谊医院疼痛二科 乌鲁木齐市,830000
临床医学
微小RNA-155CCAAT增强子结合蛋白β颈椎病大鼠软骨凋亡炎症反应
microRNA-155CCAAT-enhancer-binding protein βrat with cervical spondylo-siscartilageapoptosisinflammatory response
《医学分子生物学杂志》 2024 (004)
300-308 / 9
新疆维吾尔自治区自然科学基金(No.2022D01C326) This work was supported by a grant from the Natural Science Foundation of Xinjiang Uygur Autonomous Region(No.2022D01C326)
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