The Study On The Expression And Mechanism Of Apoptosis Regulatory Factors(miR-322,PCSK9) In Animal Model Of Neural Tube Defect

Objectives: Neural tube defects(NTDs)are the most common birth defect caused by incomplete or failed closure of the neural tubes in the early embryonic development.The incidence of NTDs is between 1 ‰ and 5 ‰,and its risk factors involve environment,biology,genetics,radiation and chemical drugs.As a complex multifactorial disease,the specific etiology of NTDs is still unknow.Gene mutation or abnormal regulatory mechanism in various signaling pathways may play an important role in the occurrence of NTDs.Apoptosis,also known as programmed cell death,plays an important role in life activities such as embryonic development,tissue and organ formation,and functional decisions.Studies have shown that the occurrence of NTDs is due to the imbalance between cell proliferation and apoptosis,which is caused by self-gene defect or dysfunction of apoptosis-related genes.In previous studies,we found that microRNA-322(miR-322)and proprotein convertase subtilisin / kexin type 9(PCSK9)were underexpressed in NTDs: In diabetes-induced NTDs,miR-322 was low-expressed and inhibited apoptosis of mouse neural stem cells induced by high glucose;in the proteomic analysis of E11 pregnant mice serum,the protein expression level of PCSK9 in NTDs group was significantly reduced compared to the control group.These findings suggest that miR-322 and PCSK9 have the potential to become new targets for early screening and treatment of NTDs,but the specific molecular mechanism involved in NTDs is unclear.Therefore,this study established an animal model of NTDs to explore the expression of miR-322 and PCSK9 in NTDs and its effect on cell apoptosis,and to clarify the mechanism of their involvement in NTDs.Methods: This study is divided into the following two parts:1.MiR-322 treatment rescues NTD formation through silencing NADPH oxidase 4(NOX4).1.1The mouse NTDs model was established using all-trans retinoic acid(ATRA)and the embryos were obtained on embryonic day 9.5(E9.5).The expression levels of miR-322 and NOX4 were detected by qRT-PCR;the expression level of NOX4 protein was detected by western blot and immunohistochemistry.1.2 Bioinformatics analysis predicted the target genes of mi R-322.C17.2 mouse neural stem cells were cultured and transfected with biotin-labeled miR-322 and negative control.Cell lysates were collected,and the RNA pull down assay was used to verify the binding of miR-322 to NOX4.1.3 C17.2 cells were transfected with indicated NOX4 luciferase reporter,together with miR-322 mimic or negative control,and the interaction between miR-322 and NOX4 was verified by double luciferase reporter analysis.Subsequently,the negative regulatory relationship between mi R-322 and NOX4 was further clarified at the cellular level.1.4 After knocking down NOX4 in C17.2 cells,the changes of relevant apoptosis markers were detected by western blot;NOX4 was overexpressed in C17.2 cells and miR-322 mimic was co-transfected at the same time,the changes of relevant apoptosis markers were detected by western blot,and the apoptosis level was observed by TUNEL assay.1.5 In whole-embryo culture,mi R-322 mimics were injected into the amniotic cavity of embryos in NTDs group.The effects of miR-322 on the reduction of apoptosis and the treatment of NTDs was evaluated by observing the tissue morphology,protein expression of NOX4 and apoptosis-related indicators and cell apoptosis level.2.The study on the expression of PCSK9 in the rat NTDs model and the mechanism of inhibition of PCSK9 causing NTDs.2.1 ATRA was used to establish rat NTDs model,and spinal cord tissue were taken at E11-E20.2.2 The expression of PCSK9 on E11-E20 was detected by qRT-PCR and western blot.2.3 Blood concentration and Western Blot were used to evaluate the effect of PCSK9 inhibitor SBC115076.2.4 To investigate whether the combination of SBC115076 and ATRA can increase the incidence of NTDs or increase the teratogenic sensitivity.2.5 PCSK9 knockout mice were used to verify that inhibition of PCSK9 can increase the teratogenic sensitivity of embryos to ATRA.2.6 After PCSK9 was inhibited,Western blot was used to verify the expression changes of caspase3,Bcl2 and Bax in C17.2 cell and spinal cord tissues,to clarify the relationship between PCSK9 and apoptosis.2.7 Lipid composition in serum samples of pregnant rat and embryonic spinal cord were analyzed by lipidomics.Results: 1.MiR-322 treatment rescues neural tube defect formation through silencing NADPH oxidase 4(NOX4).1.1 MiR-322 expression was down-regulated in ATRA-induced NTDs mouse models.In contrast,the protein expression level of NOX4 was significantly up-regulated,but the mRNA level was not changed.1.2 NOX4 is a potential target gene for miR-322,and their interaction is achieved through a binding site present on the 3'-untranslated region(3'-UTR)of NOX4 mRNA.When miR-322 was overexpressed,the protein expression level of NOX4 decreased correspondingly,but the mRNA level did not change significantly.When miR-322 was inhibited,the protein expression level of NOX4 increased significantly,but the mRNA level did not change.1.3 In C17.2 cells,NOX4 knockdown resulted in decreased expression of pro-apoptotic proteins caspase3 and Bax,while increased expression of anti-apoptotic protein bcl-2.Conversely,overexpression of NOX4 led to increased expression of caspase3 and Bax,decreased expression of bcl-2 and increased apoptosis.On this basis,transfection with miR-322 mimic can reverse the changes of apoptosis-related proteins caused by overexpression of NOX4 and reduce the apoptosis.1.4 In whole embryo culture,the expression of NOX4 protein in embryos was significantly reduced after treatment with miR-322 mimic.At the same time,Bcl-2 expression level was increased,caspase3 and Bax expression were down-regulated,and apoptosis was decreased.2.The study on the expression of PCSK9 in the rat NTDs model and the mechanism of inhibition of PCSK9 causing NTDs.2.1 On E11-E20,the mRNA and protein levels of PCSK9 in the NTDs group were significantly reduced compared with the control group.2.2 SBC115076 inhibited the expression of PCSK9 in rats.SBC115076 in combination with ATRA can increase the rate of embryo malformation.Consistent results were obtained in PCSK9 knockout mice.2.3 Inhibition of PCSK9 increased the expression of proapoptotic proteins caspase3 and Bax and decreased the expression of anti-apoptotic protein Bcl-2 in C17.2 cell and embryonic spinal cord.Conclusions: 1.NOX4 is a new target of miR-322 and interacts with miR-322 through the binding site at the 3 'UTR of NOX4 mRNA.2.Reduced expression of mir-322 can increase the expression level of NOX4 protein and induce apoptosis,which eventually leads to NTDs.3.The therapeutic effect of mir-322 on the apoptosis of NTDs embryos may become a new target for the treatment of NTDs.4.The expression of PCSK9 remained low during the development of NTD spinal cord tissue.5.Inhibition of PCSK9 increased the teratogenic sensitivity of the embryo to ATRA.6.Inhibition of PCSK9 can promote apoptosis by up-regulating Cleaved-Caspase3 and Bax/ Bcl-2,and then participate in the occurrence of NTDs.