lncRNA HOTAIR促椎间盘髓核细胞凋亡的机制研究  

Study on mechanism of lncRNA HOTAIR promoting apoptosis of intervertebral disc nucleus pulposus cells

作　　者：艾力夏提·艾热提 马志刚[1] 李洪伟[1] Ailixiati Aireti;MA Zhi-gang;LI Hong-wei(First Department of Spine,Orthopedic Center,People's Hospital of Xinjiang Uygur Autonomous Region,Urumqi Xinjiang 830000,China)

机构地区：[1]新疆维吾尔自治区人民医院骨科中心脊柱一科,新疆乌鲁木齐830000

出　　处：《局解手术学杂志》2023年第9期764-770,共7页Journal of Regional Anatomy and Operative Surgery

基　　金：新疆维吾尔自治区自然科学基金(2021D01C138)。

摘　　要：目的研究长链非编码RNA(lncRNA)HOX转录反义RNA(HOTAIR)对椎间盘髓核细胞凋亡的调控作用。方法培养人椎间盘髓核细胞系(HNPC),并将HNPC细胞随机分为对照组(Ctrl组)、晚期糖基化终末产物(AGE)组(AGE组)、微小RNA(microRNA,miR)-22的拟似物组(mimic组)、mimic的阴性对照组(mimic-NC组)、lncRNA HOTAIR过表达组(HOTAIR组)、过表达质粒的空载体组(Vector组)、HOTAIR联合mimic处理组(HOTAIR+mimic组)、HOTAIR联合核苷酸结合寡聚结构域样受体家族Pyrin域蛋白3(NLRP3)的抑制剂mcc950处理组(HOTAIR+mcc950组)。qRT-PCR法检测miR-22和lncRNA HOTAIR的表达。双荧光素酶报告基因实验检测lncRNA HOTAIR和miR-22以及miR-22和NLRP3的关系。Western blot检测NLRP3炎症小体相关蛋白的表达。流式细胞术和Hoechst 33342染色检测HNPC细胞的凋亡水平。结果与Ctrl组比较,AGE组lncRNA HOTAIR及NLRP3、Caspase1、含半胱天冬氨酸蛋白酶激活和招募结构域结构的凋亡相关斑点样蛋白(ASC)均上调,细胞凋亡增加,miR-22表达下调,差异均有统计学意义(P<0.05);lncRNA HOTAIR可以和miR-22的碱基序列结合,且miR-22能和NLRP33'UTR的碱基序列结合。与Vector组比较,HOTAIR组的lncRNA HOTAIR及NLRP3、Caspase1、ASC蛋白表达上调,细胞凋亡增加,miR-22表达下调,差异均有统计学意义(P<0.05)。与HOTAIR组比较,HOTAIR+mimic组的miR-22表达上调,NLRP3、Caspase1、ASC蛋白表达均下调,细胞凋亡数减少,差异均有统计学意义(P<0.05)。与HOTAIR组比较,HOTAIR+mcc950组的lncRNA HOTAIR和miR-22表达差异不显著(P>0.05),NLRP3、Caspase1、ASC蛋白表达下调,细胞凋亡数减少,差异均有统计学意义(P<0.05)。结论lncRNA HOTAIR通过靶向调控miR-22激活NLRP3炎症小体促进椎间盘髓核细胞凋亡。Objective To study the regulatory effect of long non-coding RNA(lncRNA)HOX transcript antisense intergenic RNA(HOTAIR)on the apoptosis of nucleus pulposus cells of intervertebral disc.Methods Human intervertebral disc nucleus pulposus cell line(HNPC)was cultured and randomly divided into control(Ctrl)group,advanced glycation end products(AGE)group(AGE group),microRNA(miR)-22 mimic group(mimic group),mimic negative control group(mimic-NC group),lncRNA HOTAIR overexpression group(HOTAIR group),empty vector of overexpression plasmid group(Vector group),HOTAIR combined with mimic treatment group(HOTAIR+mimic group),and HOTAIR combined with the inhibitor of nucleotide-binding oligomerization domain-like receptor family pyrin domaincontaining protein 3(NLRP3)mcc950 group(HOTAIR+mcc950 group).The expression of miR-22 and lncRNA HOTAIR was detected by qRT-PCR.The relationship between lncRNA HOTAIR and miR-22,as well as miR-22 and NLRP3,was detected by dual luciferase reporter gene assay.The expression of NLRP3 inflammasome-related proteins was detected by Western blot.And the apoptosis of HNPC cells was detected by flow cytometry and Hoechst 33342 staining.Results Compared with the Ctrl group,the expression of lncRNA HOTAIR,NLRP3,Caspase1,and apoptosis-associated speck-like protein containing a caspase activation and recruitment domain(ASC)protein was up-regulated,cell apoptosis was increased,and the expression of miR-22 was down-regulated in AGE group,the differences were significant(P<0.05).In addition,lncRNA HOTAIR could bind to the base sequence of miR-22,and miR-22 could bind to the base sequence of NLRP33'UTR.Compared with the Vector group,the expression of lncRNA HOTAIR,NLRP3,Caspase1,and ASC protein was up-regulated,cell apoptosis was increased,and the expression of miR-22 was down-regulated in the HOTAIR group,the differences were significant(P<0.05).Compared with the HOTAIR group,the expression of miR-22 was up-regulated,and the protien expression of NLRP3,Caspase1,and ASC was down-regulated,and cell apoptosis

关 键 词：长链非编码RNA HOX转录反义RNA miRNA-22 核苷酸结合寡聚结构域样受体家族Pyrin域蛋白3 椎间盘髓核细胞 凋亡 

分 类 号：R681.53[医药卫生—骨科学]