流体剪切力下微小RNA-222调节人髓核细胞c-fos蛋白的表达解析

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To discuss the effect of microRNA一222
stress
Weiguo,Email:liang-
【A】瞻岫_d】
vided into four
Objective
s惦cells with flow shear
condition.Methods
on the c—Fos in nucleus pulpo- Nucleus pdposus cells(NPC)were randomized di.
科(叶冬平、姚依村、梁伟国)
通信作者:叶冬平,Email:yedongpin9927@126.corn;梁伟国,Email:liangweiguol011@
126.corn DOI:10.3760/cma.i.issn.1001-9030.2016.04.058
【摘要】
的NPC),在12
目的探讨流体剪切力作用下微小RNA(microRNA)-222调节人原代髓核细胞c—fos
affect the leveI of c—fos in human nucleus plllposm ceils with flow shear stress Miao Haixiong,Ye Dongping,Yao Yicun,Liang Weig∞ Department of Orthopaedics,the First Clinical Medical College of Jinan University,Guangzhou 510220,
222组65.67 4-9.94)、
c.fos蛋白表达降低(对照组754.67 4-37.44,minic 222组730.17 4-21.75)。抑制microRNA-222,流 体剪切力加载后,与流体剪切力加载前比较,髓核细胞MEK5蛋白表达降低(对照组268.83 31.78,inhibitor 222组176.83 4-20.88),p-MEK5(对照组369.00 30.86)、p-ERK5(对照组85.67
flow
shear stress,expression of MEK5
in
nucleus
pulposus cells of
decreased(blank
controller
controller
477.00±
group 268.83±31.78),expression P—MEl(5(blank 226.50±25.55。control 369.00±22.15),P—ERK5(blank controller 48.17±5.53,control group 85.67±13.32)and c—fos increased(blank controller 646.67±32.59,control group 754.67±37.“).When microRNA一222 was over expressed and flow shear stress was loaded.expression of MEK5 in nucleus pul. posus cells increased(control group 268.83±31.78,minic 222 group 374.50±17.21),expression of P—h们溺(control group 369.00±22.15,minic 222 group 317.50±32.67),P—ERK5(control group 85.67±13.32,minie 222 group 65.67 4-9.94)and c—fos decreased(control group 754.67±37.44. minic 222 group 730.17 4-_21.75.W1他n microRNA一222 was inhibited and flow shear stress w船loaded. expression of MEK5 in nudeus pulposus cells decreased(control group 754.67 4-37.44.inhibitor 222 of P—MEK5(control group 369.00±22.15,inhibitor 222 group lgoup 730.17±21.75),expression 453.00±30.86),P—ERK5(control group 85.67±13.32,inhibitor 222 group 109.17±10.30)and c—fos(control group 754.67±37.44,inhibitor 222 group 850.33 4-33.49)increased.Expression of c—f缸 mRNA(blank controller 545.00±11.75,control group 562.17±28.90,Iflinic 222 group 5柏.17±5.53。 inhibitor 222 group 545.33 4-35.64)and ERK5 protein level(blank controller 328.33 4-25.87.control group 309.83±25.31,minic 222 group 314.17±24.00,inhibitor 222 group 309.33±29.66)had no signif- Flow shear stress inhibited microRNA一222 activity。 icant cha,vces in all groups(P>o.05).Com|uslon the level of c—fos via focal adhesion up—regulated kinase(FAK)一ERK5 which was the key signal pathway
and inhibitot-microRNA一222 respectively.The mRNA expression of c—Fos and microRNA一222 level were examined tll】舢咖reverse transcriptase—polymerase chain reaction(RT—PER).Results After loading
载后,与流体剪切力加载前比较,髓核细胞MEK5蛋白表达降低(空白对照组477.00±25.28,对照 组268.83±31.78),p-MEK5(空白对照组226.50 4-25.55,对照组369.00±22.15)、p-ERK5(空白对 照组48.17±5.53,对照组85.67±13.32)、c.fos蛋白表达升高(空白对照组646.67±32.59,对照组
754.67
4-37.44)。过表达microRNA-222,流体剪切力加载后,与流体剪切力加载前比较,髓核细胞
4-31.78,minic
MEK5蛋白表达升高(对照组268.83
22.15.minic
222组374.50±17.21),p-MEK5(对照组369.00
4-13.32,minic
4-
222组317.50 4-32.67)、p-ERK5(对照组85.67
生堡塞堕处型苤蠢垫!!生垒旦筮!!鲞墓垒翅垦!垫』垦!E!!垡:垒世!!Q!!:y尘.!!。№:堡
・1059・
・实验研究・
流体剪切力下微小RNA一222调节人髓核
细胞c—fOs蛋白的表达
缪海雄叶冬平姚依村梁伟国
作者单位:510220广州,暨南大学第一临床医学院骨科(缪海雄、叶冬平、姚依村、 梁伟国);516003惠州市第一人民医院骨科(缪海雄);510220广州市红十字会医院骨
P—ndtogen extracellular kinase ring(WB).’11圮expression of microRNA一222 were
5(衄K5),皿l(5,ERK5
oveY
sip
regulated kinase
and
c—Fos were examined
or
under regulated throu小mimic micreRNA一222
510220,吼ina(ye
DP,Yao YC,Hang WG)
万方数据
生堡塞垦处叠塞盍兰坐生璺旦筮塑鲞筮垒期堡蝤!』啦墨蚺,垒画!垫!!,!尘:箜,盟!:兰
。啪霸弘m击喈枷or:Ire
weiguolDJl@126.coin
D伽咖,Email:yedongpin9927@126.corn;Liang
groups(n=24)such group).Protein
as:blank control
group(NPC
without any
pre—treatment,u=6), 5(ERK5), throu小Western blot-
micreRNA一222 normal,over and silencing expressions(NPC witll 12 dyn/cm2×45 min flow shear stress,n=6/per expressions of P—exuaeelhlar
水平。方法分组:原代人髓核细胞(NPC,rg=24)随机平均分为4组:空白对照组(未经任何处理
dyn/cm2×45
min流体剪切力加载条件下microRNA一222未干扰组(Control组)、
micmRNA一222过表达组(minic 222组)和microRNA-222沉默组(inhibitor 222组)。Western blot检测 各组黏着斑激酶(FAK)一细胞外信号调节激酶5(ERK5)信号通路中磷酸化ERK5(p-ERK5)、磷酸化 丝裂原细胞外激酶5(p-MEK5)、丝裂原细胞外激酶5(MEK5)及其信号通路下游c—fos的蛋白表达。 通过mimic RNA一222及inhibitor RNA.222对microRNA-222进行过表达或沉默,通过反转录一聚合酶 链反应(RT—PCR)验证过表达或沉默效率,并检测各组c-fos的mRNA水平。结果 流体剪切力加