MicroRNA-222 carried by plasma-derived exosomes as an earlier biomarker to alert the abnormal phosphorylation of Tau caused by shRNA-PCSK9 and its correlation with Aβ1-42Ab.

Abstract The clinical adverse reaction monitoring data of Human subtilisin converting enzyme 9 gene (proprotein converting enzyme subtilisin/kexin type 9, PCSK9) inhibitors showed that there were neurocognitive adverse events. In order to early warning of the potential danger of PCSK9 inhibitors to cognitive impairment in the process of regulating lipid homeostasis, this study aimed to explore the microscopic effects of plasma exosomes. MicroRNA-222 (miR-222) as an early warning marker for shRNA-PCSK9-induced cognitive impairment. The hypercholesterolemia mouse model (high-fat diet, HFD-model group) was prepared by feeding with high-fat diet. The HFD-models were then divided into shRNA control group (HFD-shRNA-control group) and shRNA PCSK9 group (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) and immunohistochemistry (IHC). Tau protein and phosphorylation in brain tissue were observed by IHC. Western blot(WB)was used to detect Tau protein and P-Tau protein. Serum amyloid Aβ1-42Ab levels were determined by enzyme-linked immunosorbent assay (ELISA). The kits extract plasma exosomes step by step, identify the exosome morphology by negative staining electron microscopy, and determine the size of exosomes by NTA technology. RT-PCR technique was used to detect the expression level of miR-222 carried in plasma exosomes. The HFD-model model was prepared by feeding a high-fat diet for 13 weeks, and the serum total cholesterol (TC) and low-density lipoprotein (LDL-C) contents were significantly increased. At the same time, the expression of PCSK9 mRNA in the brain tissue of HFD-model mice 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 have occurred. However, at this time, serum Aβ1-42Ab has not been significantly increased, and it has not yet been of significance for the diagnosis of cognitive impairment. The microRNA in plasma exosomes was extracted, and RT-PCR results showed that the expression of miR-222 carried in the exosomes of the HFD-shRNA-PCSK9 group was significantly lower than that of the HFD-shRNA-control group. At the same time, MiR-222 levels in plasma-derived exosomes were inversely correlated with Aβ1-42Ab in serum. MiR-222 carried by plasma-derived exosomes provides an early warning marker for shRNA-PCSK9-induced cognitive impairment.