流体剪切力下微小RNA-222调节人髓核细胞c-fos蛋白的表达

MicroRNA-222 affect the level of c-fos in human nucleus pulposus cells with flow shear stress

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水平.方法 分组：原代人髓核细胞（NPC,n=24）随机平均分为4组：空白对照组（未经任何处理的NPC）,在12 dyn/cm^2×45 min流体剪切力加载条件下microRNA-222未干扰组（Control组）、microRNA-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）-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-222及inhibitor RNA-222对microRNA-222进行过表达或沉默,通过反转录-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±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±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MEK5蛋白表达升高（对照组268.83±31.78,minic 222组374.50±17.21）,p-MEK5（对照组369.00±22.15,minic 222组317.50±32.67）、p-ERK5（对照组85.67±13.32,minic 222组65.67±9.94）、c-fos蛋白表达降低（对照组754.67±37.44,minic 222组730.17±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±20.88）,p-MEK5（对照组369.00±22.15,inhibitor 222组453.00±30.86）、p-ERK5（对照组85.67±13.32,inhibitor 222组109.17±10.30）、c-fos蛋白表达升高（对照组754.67±37.44,inhibitor 222组850.33±33.49）,c-fos mRNA表达量（空白对照组545.00±11.75,对照组562.17±28.90,minic 222组548.17±5.53,inhibitor 222组545.33±35.64）、ERK5蛋白表达量（空白对照组328.33±25.87,实验对照组309.83±25.31,minic 222组314.17±24.00,inhibitor 222组309.33±29.66）在各组差异无统计学意义（P＞0.05）.结论 流体剪切力通过抑制microRNA-222促进c-fos蛋白表达并上调髓核细胞骨架关键通路FAK-ERK5活性,加速髓核细胞退变.

Objective To discuss the effect of microRNA-222 on the c-Fos in nucleus pulposus cells with flow shear stress condition.Methods Nucleus pulposus cells （NPC） were randomized divided into four groups （n =24） such as：blank control group （NPC without any pre-treatment,n =6）,microRNA-222 nonnal,over and silencing expressions （NPC with 12 dyn/cm^2× 45 min flow shear stress,n =6/per group）.Protein expressions of p-extracellular signal regulated kinase 5 （ERK5）,p-mitogen extracellular kirase 5 （MEK5）,MEK5,ERK5 and c-Fos were examined through Western blotting （WB）.The expression of microRNA-222 were over or under regulated through mimic microRNA-222 and inhibitor microRNA-222 respectively.The mRNA expression of c-Fos and microRNA-222 level were examined through reverse transcriptase-polymerase chain reaction （RT-PCR）.Results After loading flow shear stress,expression of MEK5 in nucleus pulposus cells decreased （blank controller 477.00 ± 25.28,control group 268.83 ± 31.78）,expression of p-MEK5 （blank controller 226.50 ± 25.55,control group 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 ± 37.44）.When microRNA-222 was over expressed and flow shear stress was loaded,expression of MEK5 in nucleus pulposus cells increased（control group 268.83 ± 31.78,minic 222 group 374.50 ± 17.21）,expression of p-MEK5 （control group 369.00 ± 22.15,minic 222 group 317.50 ± 32.67）,p-ERK5 （control group 85.67 ± 13.32,minic 222 group 65.67 ± 9.94） and c-fos decreased（control group 754.67 ± 37.44,minic 222 group 730.17 ± 21.75.When microRNA-222 was inhibited and flow shear stress was loaded,expression of MEK5 in nucleus pulposus cells decreased （control group 754.67 ± 37.44,inhibitor 222 group 730.17 ± 21.75）,expression of p-MEK5 （control group 369.00 ± 22.15,inhibitor 222 group 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 ± 33.49） increased.Expression of c-fos mRNA （blank controller 545.00 ± 11.75,control group 562.17 ± 28.90,minic 222 group 548.17 ± 5.53,inhibitor 222 group 545.33 ± 35.64） and ERK5 protein level （blank controller 328.33 ± 25.87,control group 309.83 ± 25.31,minic 222 group 314.17 ± 24.00,inhibitor 222 group 309.33 ± 29.66） had no significant changes in all groups （P 〉 0.05）.Conclusion Flow shear stress inhibited microRNA-222 activity,up-regulated the level of c-fos via focal adhesion kinase （FAK）-ERK5 which was the key signal pathway for nucleus pulposus cells cytoskeleton and promoted NPC regression.