Lnc-NEAT1吸附miR-1224激活NF-κB通路促进SCI大鼠骨质流失

Lnc-NEAT1 adsorbs miR-1224 and activates NF-κB pathway to promote bone loss in SCI rats

目的探讨长链非编码RNA富含核的丰富转录物1(Lnc-NEAT1)对脊髓损伤(SCI)大鼠骨质流失的调控机制。方法64只Wistar大鼠随机分为SCI组[脊髓横断(T10)全切术建立SCI,n=56]和假手术组(sham组)(仅切开皮肤即缝合,n=8)。随机选择48只SCI组的大鼠于术后10 d随机分为6组进行药物注射,包括SCI+Lnc-NEAT1过表达载体组(SCI+pcDNA-NEAT1组)、SCI+pcDNA阴性对照组(SCI+pcDNA-NC组)、SCI+pcDNA-NEAT1+miR-1224拟似物组(SCI+pcDNA-NEAT1+miR-1224-mimic组)、SCI+pcDNA-NEAT1+mimic阴性对照组(SCI+pcDNA-NEAT1+mimic-NC组)、SCI+pcDNA-NEAT1+miR-1224-mimic+NF-κB抑制剂QNZ组(SCI+pcDNA-NEAT1+miR-1224-mimic+QNZ组)、SCI+pcDNA-NEAT1+miR-1224-mimic+DMSO组。治疗10 d后通过micro-CT检测骨体积分数(BV/TV)、骨小梁数目(Tb.N)、骨小梁间距(Tb.Sp)、骨小梁厚度(Tb.Th)。qPCR法检测Lnc-NEAT1、miR-1224、肿瘤坏死因子(TNF)-α、白细胞介素(IL)-6、IL-1β、干扰素(IFN)-γ、可溶性白细胞介素2受体(sIL-2R)的表达。Western blot测NF-κB、磷酸化的NF-κB p65(p-NF-κB p65)的表达。荧光素酶基因报告实验检测Lnc-NEAT1和miR-1224的直接调控关系。结果与sham组比,SCI组的miR-1224表达水平降低(P<0.05),Lnc-NEAT1、NF-κB p65、p-NF-κB p65的表达量上调(均P<0.05)。与SCI+pcDNA-NC组相比,SCI+pcDNA-NEAT1组的miR-1224表达水平和BV/TV、Tb.N、Tb.Th的值都降低(均P<0.05),而Tb.Sp的值和Lnc-NEAT1、TNF-α、IL-6、IL-1β、IFN-γ、sIL-2R、NF-κB p65、p-NF-κB p65的表达量上调(均P<0.05)。SCI+pcDNA-NEAT1+miR-1224-mimic组抑制了SCI+pcDNA-NEAT1组的上述效果(均P<0.05),但不影响Lnc-NEAT1的水平。SCI+pcDNA-NEAT1+miR-1224-mimic+QNZ组进一步增加了SCI+pcDNA-NEAT1+miR-1224-mimic组的治疗效果,使BV/TV、Tb.N、Tb.Th的值都被上调(均P<0.05),而Tb.Sp的值和TNF-α、IL-6、IL-1β、IFN-γ、sIL-2R、NF-κB p65、p-NF-κB p65的表达量都被抑制(均P<0.05)。另外证实,Lnc-NEAT1通过“分子海绵”机制直接抑制miR-1224的表达。结论Lnc-NEAT1通过抑制miR-1224激活NF-κB信号抑制SCI大鼠的骨丢失并减轻免疫炎症反应。

Objective To investigate the regulatory mechanism of long non-coding RNA NEAT1(Lnc-NEAT1)on bone loss in rats with spinal cord injury(SCI).Methods Sixty-four Wistar rats were randomly divided into SCI group SCI was established by total spinal cord transection(T10,n=56)and sham group(skin incision and su-ture only,sham group,n=8).A total of 48 SCI rats were randomly divided into 6 groups for drug injection 10 days after operation,including SCI+Lnc-NEATI overexpression vector(SCI+pcDNA-NEATI)group,SCI+pcDNA negative control(SCI+pcDNA-NC)group,SCI+pcDNA-NEAT1+miR-1224 mimic(SCI+pcDNA-NEAT1+miR-1224-mimic)group,SCI+pcDNA-NEAT1+mimic negative control(SCI+pcDNA-NEATI+mimic-NC)group,SCI+pcDNA-NEAT1+miR-1224-mimic+NF-κB inhibitor QNZ(SCI+pcDNA-NEAT1+miR-1224-mimic+QNZ)group,and SCI+pcDNA-NEAT1+miR-1224-mimic+DMSO group.After 10 days of treatment,bone vol-ume fraction(BV/TV),trabecular number(TB.N),trabecular spacing(TB.sp)and trabecular thickness(Tb.Th)were detected by micro-CT.The expressions of Lnc-NEAT1,miR-1224,tumor necrosis factor(TNF)-α,interleukin-6(IL)-6,IL-1β,interferon(IFN)-and soluble interleukin-2 receptor(sIL-2R)were detected by qPCR.The expressions of NF-κB and phosphorylated NF-κB p65(p-NF-κB p65)were detected by Western blot.Luciferase gene reporter assay was used to detect the direct regulatory correlations between Lnc-NEAT1 and miR-1224.Results Compared with sham group,the expression level of miR-1224 in SCI group was significantly reduced(P<0.05);and the expression levels of Lnc-NEAT1,NF-κB p65 and p-NF-κB p65 were significantly up-regulated(all P<0.05).Compared with the SCI+pcDNA-NC group,the expression level of miR-1224 and the values of BV/TV,TB.N and TB.Th in the SCI+pcDNA-NEAT1 group were significantly reduced(all P<0.05).The value of Tb.Sp and the expression levels of Lnc-NEAT1,TNF-α,IL-6,IL-1β,IFN-,sIL-2R,NF-κB p65 and p-NF-κB p65 were up-regulated(all P<0.05).The SCI+pcDNA-NEAT1+miR-1224-mimic group inhibited the above effects of the SCI+pcDNA-NEAT1 group(all P<0.05),but did not affect the level of Lnc-NEAT1.In the SCI+pcDNA-NEAT1+miR-1224-mimic+QNZ group,the therapeutic effect of SCI+pcDNA-NEAT1+miR-1224-mimic group was further increased,and the values of BV/TV,TB.N and TB.th were up-regulated(all P<0.05).However,the value of Tb.Sp and the expression levels of TNF-α,IL-6,IL-1β,IFN-,sIL-2R,NF-κB p65 and p-NF-κB p65 were inhibited(all P<0.05).In addition,Lnc-NEAT1 directly inhibited the expression of miR-1224 through a molecular sponge mechanism.Conclusion Lnc-NEAT1 inhibits bone loss and attenuates immune inflammation in SCI rats by inhibiting miR-1224 and activating NF-κB signaling.