过表达miR-21对人髓核细胞增殖的影响及机制研究  

MiR-21 promotes proliferation of human nucleus pulposus cells through PTEN/Akt signaling pathway

作　　者：艾则孜·艾海提[1] 安梦宇 AI Ze-zi·AI Hai-ti;AN Meng-yu(The People's Hospital of Xinjiang Uygur A utonomous Region,Urumqi 830001,Xinjiang,China)

机构地区：[1]新疆维吾尔自治区人民医院,新疆乌鲁木齐830001

出　　处：《生物医学工程与临床》2022年第5期615-620,共6页Biomedical Engineering and Clinical Medicine

摘　　要：目的 探讨miR-21对人髓核(NP)细胞增殖的影响及可能机制。方法 选取人NP细胞。选择30例腰椎间盘突出患者(其中男性21例,女性9例;年龄29~56岁,平均年龄46.18岁)及4例特发性脊柱侧凸患者(其中男性3例,女性1例;年龄18~22岁,平均年龄20.38岁)的NP组织(正常组)为研究对象。对NP细胞进行转染,分为Untreated组(未进行任何处理)、Scramble组(转染miR scramble)、miR-21组(转染miR-21-mimic)。各组分别加入二甲基亚砜(DMSO)、丝氨酸/苏氨酸蛋白激酶(Akt)抑制剂Ly294002。采用实时荧光定量聚合酶链式反应检测细胞、组织中miR-21、PTEN-mRNA表达水平;四甲基偶氮唑盐(MTT)检测细胞增殖能力;Western blot检测磷酸酶和张力蛋白同源物(PTEN)、CyclinD1、Akt、p-Akt含量;生物信息预测miR-21与PTEN的靶向作用关系,荧光素酶实验验证。结果 椎间盘退行性变NP组织miR-21 mRNA水平(2.62±0.12)高于正常NP组织(0.34±0.07)(P <0.01)。72 h、96 h时,miR-21组细胞活力、CyclinD1蛋白水平明显高于Untreated组、Scramble组(P <0.05)。miR-21-mimic+WT-PTEN组细胞荧光素酶活性(0.31±0.09)低于miR scramble+WT-PTEN组(0.84±0.11)(P <0.05)。miR-21组细胞PTEN蛋白、mRNA表达水平明显低于Untreated组、Scramble组(P <0.05)。miR-21组细胞p-AKT蛋白/Akt蛋白明显高于Untreated组、Scramble组(P <0.01)。72 h和96 h时,Ly294002+miR-21组细胞活力、CyclinD1蛋白表达水平明显低于DMSO+miR-21组(P <0.01)。结论 miR-21在椎间盘退行性变NP组织中高表达,可通过PTEN/Akt通路促进NP细胞增殖。Objective To explore the influence of miR-21 on proliferation of nucleus pulposus(NP) cells and the underlying mechanism. Methods Human NP cells and tissues were obtained from 30 patients with lumbar disc herniation(21 males and9 females, aged 29-56 years old with mean age of 46.18 years old) and 4 patients with idiopathic scoliosis(3 males and 1female, aged 18-22 years old with mean age of 20.38 years old). The NP cells were transfected and grouped into Untreated group(untreated NP cells), Scramble group(miR-scramble) and miR-21 group(miR-21-mimic). Dimethylsulfoxide(DMSO) and serine/threonine protein kinase(Akt) inhibitor LY294002 were used. The levels of miR-21, phosphatase and tensinhomolog(PTEN)-mRNA were detected by real time polymerase chain reaction(PCR), and methyl thiazolyl tetrazolium(MTT) was used to analyze cell proliferation ability. The expression levels of PTEN, CyclinD1, Akt and p-Akt protein were detected by Western blot, the target effects of miR-21 and PTEN were predicted by bioinformatics and validated by luciferase assay. Results The level of miR-21 in degenerative NP(2.62 ± 0.12) was higher than that in normal NP(0.34 ± 0.07)(P < 0.01). At 72-hour and96-hour, the cell viability, expression of CyclinD1 protein of miR-21 group were significantly higher than those of Untreated group and Scramble group(P < 0.05). Compared with miR scramble + WT-PTEN group(0.84 ± 0.11), the luciferase activity of miR-21-mimic + WT-PTEN group(0.31 ± 0.09) was significantly decreased(P < 0.01). The level of PTEN protein and mRNA expression in miR-21 group were lower than those of Untreated guoup and Scramble group( P < 0.05). p-Akt protein/Akt protein in miR-21 group was significantly higher than that in Untreated group and Scramble group(P < 0.01). At 72-hour and96-hour, the cell viability and expression of CyclinD1 protein in LY294002 + miR-21 group were significantly lower than those in DMSO + miR-21 group(P < 0.01). Conclusion It is demonstrated that miR-21 is highly expressed in NP tissues of intervertebr

关 键 词：MIR-21 磷酸酶和张力蛋白同源物(PTEN) 丝氨酸/苏氨酸蛋白激酶(Akt) 髓核细胞 

分 类 号：R681.53[医药卫生—骨科学] Q754[医药卫生—外科学]