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 ex-pression

唐家国 ¹李晨芳 ²左斌 ¹何精选 ¹夏晓枫 ¹王鹏 ¹邹凯 ¹娄文杰¹

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作者信息

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
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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(Collagen Ⅱ)免疫荧光染色对其鉴定.NP细胞分成Ctrl组、白细胞介素-1 β(interleukin-1 β,IL-1 β)组、IL-1β+NC inhibitor组、IL-1β+miR-455-3p inhibitor组、IL-1β+miR-455-3p inhibitor+si-NC组、IL-1 β+miR-455-3p inhibitor+si-sirt1 组,除Ctrl组外的五组细胞均使用 10 ng/mL的IL-1 β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Collagen Ⅱ、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-1 β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、Collagen Ⅱ、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-1 β诱导的NP细胞凋亡并促进NP细胞增殖.

Abstract

Objective To investigate the influence of miR-455-3p on the proliferation and apoptosis of rat intervertebral disc nu-cleus pulposus(NP)cells by regulating the expression of silent information regulator 2 homolog 1(sirt1).Methods NP cells were i-solated 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 inhib-itor 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-1βwas 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 tar-geting relationship between miR-455-3p and sirt1.Results Rat in-tervertebral disc NP cells were successfully isolated.Compared with the IL-1β group and IL-1β+NC inhibitor group,miR-455-3p expres-sion,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 di-rectly 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-in-duced NP cell apoptosis and promote NP cell proliferation by targeting sirt1.

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

Key words

miR-455-3p/silent mating type information regulation 2 homolog 1/rats/intervertebral disc nucleus pulposus cells/proliferation/apoptosis

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基金项目

湖北省卫生健康科研基金资助项目(WJ2019H388)

武汉市卫生健康科研基金资助项目(WX19Q41)

出版年

2024

颈腰痛杂志

安徽医科大学

颈腰痛杂志

CSTPCD

影响因子：1.006

ISSN：1005-7234

参考文献量15

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