The Regulatory Molecular Mechanism Study Of MiR-200c-3p On Alzheimer’s Disease

miR-200c-3p is the member of the miR-200 family and it was found to be up-regulated in Huntington’s disease and cerebral ischemia mouse brain tissues, which strongly suggest that miR-200c-3p may play an important role in the CNS development and the aging process.CREB1 is a kind of key nuclear transcription factor that plays a significant role in memory formation.Bioinformatics analysis showed that there is a possible target of miR-200c-3p in CREB1 mRNA3’UTR sequence. At present, the role of miR-200c-3p in regulation of CREB1 has not been cleared, its function in the process of Alzheimer’s disease remains to be determined.Objectives:This study is designed to insight into the regulatory of miR-200c-3p on key nuclear transcription factor CREB1 and to reveal the regulatory molecular mechanism of miR-200c-3p on AD by detecting expression of miR-200c-3p and CREB1 differences in hippocampus between AD and control mice,analysing the relationship between miR-200c-3p and CREB1 expression in hippocampus during AD process.Methods:Kunming mice were randomly grouped into AD model group and control group, the model group were treated with AICl3 40 mg.kg(-1).d(-1) solution i.p and D-galactose 90 mg.kg(-1).d(-1)i.p, once a day for 90 days, while the control group were treated with saline water. After injection of D-galactose and AICl3 for 90 days, novel object recognition test was performed to test the differences of recognition and memory ability between AD and control mice. The expression of miR-200c-3p in the hippocampus of AD model group and control group mice were detected by Quantitative Real-time PCR. Then, the key nuclear transcription factor CREB1 protein expression in hippocampus of AD and control mice was analyzed by western blot. To confirm the direct binding of miR-200c-3p to CREB1 mRNA 3’UTR region, firstly, the expression vector pGL3C-CREB1 3’UTR and pGL3-CREB1 3’UTR mutant were constructed, then,miR-200c-3p mimic and pGL3-CREB1 3’UTR (luciferase report vector containing the CREB1 mRNA 3’UTR region) or pGL3-CREB1 3’UTR mutant was transfected into HEK293 cells, luciferase and renilla signals were measured 48 h after transfection by using the Dual Luciferase Reporter Assay Kit. To further confirm the regulatory of miR-200c-3p on key nuclear transcription factor CREB1, miR-200c-3p mimic or inhibitor was transfected into the Neuro2a cells, the cells were collected at 24 h after transfection, then the CREB1 protein was analyzed by western blot.Results:(1) The weight of AD mice was significantly lower than that of control group from the 6th to 11th week during D-galactose and AlCl3 injection. (2) In the object recognition test, AD model mice showed quite less exploration time on new object (P<0.01) and significant lower discrimination index(P<0.001) compared with that of control group. This results demonstrated that the recognition and memory ability of AD mice was seriously damaged. (3) Quantitative Real-time PCR revealed that the expression of miR-200c-3p was much higher in hippocampus of AD mice than that in the control mice(P<0.01). (4) Western blot showed that CREB1 protein levels was significantly lower in hippocampus of AD mice than that in the control mice(P<0.01). (5) The luciferase activity was significantly repressed when miR-200c-3p mimic was co-transfected with pGL3C-CREB1 3’UTR (P< 0.05). The luciferase activity did not reduce notably when miR-200c-3p mimic co-transfection with pGL3C-CREB1 3’UTR mutant. The results of Luciferase activity assay demonstrated that miR-200c-3p can inhibit CREB1 protein expression by targeting its mRNA3’UTR. (6) When miR-200c-3p mimic was transfected into Neuro2a cells, the CREB1 expression of the cells was significantly repressed (P<0.01), while, the cells were transfected with miR-200c-3p inhibitor, the expression of CREB1 was significantly upregulated (P< 0.01).Conclusions:This study suggested that miR-200c-3p expression was much higher in hippocampus of the AD mice which induced by D-galactose and AlCl3 than that in the control mice, and the key nuclear transcription factor CREB1 expression in hippocampus of the AD mice was significantly repressed; miR-200c-3p is involved in regulation of AD by targeting CREB1 mRNA’s 3’UTR, inhibiting CREB1 protein expression.