Molecular Mechanisms Of MiR-656-3p Targeting ATP10A Regulating Apoptosis In Alzheimer’s Disease Model Cells

Background Alzheimer’s disease（AD）is closely related to the environment and genetic factors,but the molecular mechanism of its pathological evolution is still unclear.MicroRNAs（miRNAs）are a class of non-coding small RNA molecules,which are about 18-25 nt and widely expressed in mammals.They are highly conservative.The 3,UTR sequence,which is complementary to the downstream target gene,can inhibit or degrade the target gene mRNA and regulate the post-transcriptional level.Studies have shown that more than 70%of the mRNA can be expressed in brain tissue,regulating the growth and differentiation of nerve-related cells.Studies have confirmed that many kinds of miRNAs are abnormally expressed in the course of Alzheimer’s disease evolution,but the specific molecular mechanism is still unclear.The purpose of this study was to explore the role of miR-656-3p in Alzheimer’s disease and its related mechanisms,and to provide theoretical basis for further elucidating the molecular mechanism of Alzheimer’s disease and potential molecular targets for the development of new drugs in clinic.Part 1.Study on the Apoptosis of miR-656-3p in in Alzheimer’s Disease Model CellsObjective To explore the regulatory role of miR-656-3p in the proliferation and apoptosis of Alzheimer’s disease model cells.Methods SH-SY5Y cells,which cultured in vitro,were induced by different concentrations of Aβ_25-35（0μmol/L、5μmol/L、10μmol/L、20μmol/L）to construct cell models.SH-SY5Y cells were randomly divided into model group,NC group and miR-656-3p group.Normal cultured SH-SY5Y cells were selected as control group.MTT assay was used to detect cell proliferation activity.The expression of miR-656-3p were detected by qPCR in SH-SY5Y cells induced by different concentrations of Aβ_25-35 and at different time（0,24,48,72 h）.The expression of miR-656-3p were detected by qPCR by transfection of miR-656-3p mimics.The apoptotic rate was detected by flow cytometry.Results Aβ_25-35 significantly decreased the proliferation activity of SH-SY5Y cells in a concentration-dependent manner（P<0.05）,and significantly inhibited the expression of miR-656-3p（P<0.05）.When SH-SY5Y cells were treated with 10μmol/L Aβ_25-35 for24h,the expression of miR-656-3p changed greatly.Therefore,SH-SY5Y cells were induced by 10μmol/L Aβ_25-35 to make Alzheimer’s disease cell model.Compared with the control group,the cells in the model group significantly inhibited cell proliferation（P<0.05）and promoted cell apoptosis（P<0.05）.Overexpression of miR-656-3p promoted the proliferation of model cells and inhibited cell apoptosis（P<0.05）.conclusion The expression of miR-656-3p in Alzheimer’s disease model cells decreased.Overexpression of miR-656-3p could promote the proliferation of Alzheimer’s disease model cells and inhibit their apoptosis,thus playing a protective role in Alzheimer’s disease.Part 2.Related studies on the target genes of miR-656-3pObjective The purpose of this study was to investigate the downstream target genes of miR-656-3p regulating cell proliferation and apoptosis in Alzheimer’s disease model cells.Methods Target Scan tools online software predicted that miR-656-3p and target gene 3,-UTR pairing each other.According to the reported references,ATP10A was selected as the target gene.Dual luciferase reporter gene was used to verify the regulatory effect of miR-656-3p and ATP10A.q PCR and Western blot detected the effect of up-regulation and down-regulation of miR-656-3p on the expression of ATP10A in cells.The effects of knockdown of ATP10A on cell proliferation and apoptosis were also observed.Results Prediction results of online database showed that there were complementary phenomena between miR-656-3p and ATP10A 3,-UTR.Dual luciferase reporter gene validated that the binding sites of miR-656-3p and ATP10A.Overexpression and knockdown of miR-656-3p had no significant effect on ATP10A m RNA level,but could significantly regulate ATP10A protein level（P<0.05）;knockdown of ATP10A significantly promoted the proliferation activity of model cells and inhibited apoptosis（P<0.05）.conclusion ATP10A is the target gene of miR-656-3p.miR-656-3p negatively regulates the expression of ATP10A.knockdown of ATP10A could induces the proliferation,and inhibit apoptosis.Part 3.miR-656-3p targeting ATP10A gene regulates apoptosis in alzheimer’s disease model cellsObjective The purpose of this study was to explore the effect of miR-656-3p on apoptosis of alzheimer’s disease model cells by regulating ATP10A.Methods Co-transfected overexpression of miR-656-3p and ATP10A and knockdown of miR-656-3p and ATP10A into model cells.Western blot was used to detect the expression of ATP10A after transfection.MTT and flow cytometry were used to detect the changes of cell proliferation and apoptosis.Results Compared with NC group,overexpression of miR-656-3p significantly decreased ATP10A protein level（P<0.05）.Compared with anti-NC group,knockdown of miR-656-3p significantly increased ATP10A protein level（P< 0.05）.miR-656-3p +ATP10A group reversed the inhibitory effect of miR-656-3p on ATP10A protein level,apoptotic rate and proliferation（P<0.05）.anti-miR-656-3p+si-ATP10A reversed the effects of anti-miR-656-3p on ATP10A protein level,apoptotic rate and proliferation（P<0.05）.Conclusion miR-656-3p regulates the proliferation,apoptosis of alzheimer’s disease model cells by targeting ATP10A.