外泌体miRNA-222预警shRNA-PCSK9诱发脑Tau蛋白过度磷酸化

Exosomal miRNA-222 Alerts shRNA-PCSK9 Induction Brain Tau Hyperphosphorylation

关键词:  高胆固醇血症  PCSK9  认知障碍  miRNA-222

Key words:  hypercholesterolemia  PCSK9  cognitive impairment  miRNA-222

摘要:

目的 探究血浆外泌体携带的微小RNA-222(microRNA-222，miRNA-222)能否作为shRNA-PCSK9 诱发认知障碍提供早期预警的标志物。方法 高脂饲料(high-fat diet，HFD)喂饲法制备高胆固醇血症小鼠模型。再将模型小鼠分为HFD-shRNA空白对照组和HFD-shRNA-PCSK9组。构建shRNA-PCSK9慢病毒，经静脉注入体内，实时荧光定量(realtime polymerase chain reaction，RT-PCR)法检测PCSK9 mRNA表达。免疫组织化学(immunohistochemistry，IHC)观察脑组织Tau蛋白和磷酸化。Western blotting检测Tau蛋白和P-Tau蛋白。ELISA法测定血清淀粉样蛋白Aβ1-42Ab水平。试剂盒分步提取血浆外泌体，负染电镜技术对外泌体形态进行鉴定，纳米颗粒跟踪分析技术测定外泌体粒径。RT-PCR技术检测血浆外泌体中携带的miRNA-222表达水平。结果 HFD喂饲13周制备模型组小鼠，血清总胆固醇(total cholesterol，TC)、低密度脂蛋白(low density lipoprotein，LDL-C)含量显著升高，同时，模型组小鼠脑组织内PCSK9 mRNA表达明显升高。经shRNA-PCSK9慢病毒干扰后，PCSK9 mRNA表达抑制，同时IHC观察到shRNA-PCSK9诱发了脑组织Tau蛋白的异常表达和过度磷酸化，表明已发生神经纤维缠结的病理改变。然而，此时血清Aβ1-42Ab尚未明显升高，尚未具备诊断认知障碍的意义。将血浆外泌体中的miRNA提取，RT-PCR结果显示，HFD-shRNA-PCSK9组外泌体中携带的miRNA-222表达量与HFD-shRNA空白对照组相比显著降低。结论 血浆外泌体携带的miRNA-222可以作为shRNA-PCSK9 诱发认知障碍提供早期预警的标志物。

Abstract:

OBJECTIVE To investigate whether the microRNA-222(miRNA-222) carried by plasma exosomes can serve as an early warning marker for cognitive impairment induced by shRNA-PCSK9. METHODS The high-fat diet(HFD) was used to prepare a hypercholesterolemic mouse model group. The model group mice were divided into HFD-shRNA control group and HFD-shRNA-PCSK9 group. The shRNA-PCSK9 was constructed, injected intravenously into the body, and the expression of PCSK9 mRNA was detected by real-time PCR(RT-PCR). Tau protein and phosphorylation in brain tissue were observed by immunohistochemistry (IHC). Western blotting was used to detect Tau protein and P-Tau protein. Serum amyloid Aβ1-42Ab levels were determined by ELISA. The kits extracted plasma exosomes step by step, identify the exosome morphology by negative staining electron microscopy, and determined the size of exosomes by NTA technology. RT-PCR technique was used to detect the expression level of miRNA-222 carried in plasma exosomes. RESULTS The model mouse were prepared by feeding HFD for 13 weeks, whose total cholesterol(TC) and low-density lipoprotein(LDL-C) contents in serum were significantly increased. At the same time, the expression of PCSK9 mRNA in the brain tissue of model group was significantly increased. After shRNA-PCSK9 lentivirus interference, PCSK9 mRNA expression was inhibited, and IHC observed that shRNA-PCSK9 induced abnormal expression and hyperphosphorylation of Tau protein in brain tissue, indicating that the pathological changes of neurofibrillary tangles had occurred. However, at this time, serum Aβ1-42Ab had not been significantly increased, and it had not yet been of significance for the diagnosis of cognitive impairment. The miRNA in plasma exosomes was extracted, and RT-PCR results showed that the expression of miRNA-222 carried in the exosomes of the HFD-shRNA-PCSK9 group was significantly lower than that of the HFD-shRNA control group. CONCLUSION Plasma exosomes carried miRNA-222 provides an early warning marker for shRNA-PCSK9- induced cognitive impairment.