| PART ONE: KEN PROMOTES ADAM10 TRANSLATIONTHROUGH ADAM10 5’ UTRObjective:To investigate the effect of small molecule drug Kenpaullone(KEN)on ADAM10 expression in several types of cells,and to explore the mechanism in it.Method:1.Using Western Blot assays to observe the alteration of ADAM10 protein expression in SH-SY5 Y cells that treated with KEN,Alsterpaullone,a structural analog of KEN,and AT7519,which is also a CDK/GSK3 inhibitor.2.The expression level of ADAM10 protein in several types of cells under the treatment of KEN were detected by Western Blot assays.3.SH-SY5 Y cells were treated with time gradient and concentration gradient of KEN,and Western Blot assays were used to explore the most effective action time and concentration.4.The expression intensity of ADAM10 protein in SH-SY5 Y cells were detected by immunofluorescence technique under the treatment of KEN at effective concentration and time.5.The viability of SH-SY5 Y cells under the treatment of several concentrations of KEN were detected by CCK-8 kit.6.Western Blot assays were used to explore the effect of KEN on the expression of APP protein and secreted protein s APPα from SH-SY5Y-APP cells.7.ELISA kit was used to detect the difference of Aβ1-42 secreted from SH-SY5Y-APP cells after KEN treatment compared with the control group.8.To explore the effect of KEN on the ADAM10 m RNA expression of SH-SY5 Y cells by using q-PCR assay.9.Using Western Blot assays to observe the effect of KEN on the expression of ADAM10 could be interrupted by which reagent,the transcription inhibitor reagent Act D,the translation inhibitor reagent CHX,the proteasome inhibitor reagent MG132,the lysosomal inhibitor reagent CQ or the translation initiation factor complex competitive inhibitor reagent 4EGI-1.10.The si RNA of CDK5 or GSK3β was transfected into the cells,and Western Blot assay was used to observe whether they could interrupt the induced effect of KEN on ADAM10 translation.11.Cells were transfected with ADAM10 plasmid containing or not containing the 5’UTR fragment,the targeted effective fragment of KEN was detected by Western Blot assay.12.The sequence containing the promoter and 5’UTR fragment of ADAM10 was cloned into the firefly luciferase reporter vector p GL4.17,subsequently cells were transfected with corresponding plasmid.Using luciferase assays to detect fluorescence intensity of each group.Result:1.SH-SY5 Y cells were exposed to different concentrations of three reagents(KEN,Alsterpaullone,AT7519),only KEN significantly increased the protein expression of ADAM10,compared with the control(**p <0.01).2.Under the treatment of the time gradient of KEN(0-48 hours)in SH-SY5 Y cells,the ADAM10 protein expression increased most significantly at 36 hours(** p < 0.01).3.The expression of ADAM10 protein in human embryonic kidney HEK293 cells,mouse hippocampal HT22 cells,and mouse primary neurons increased under the treatment of different concentrations(0-2.0μM)of KEN(** p < 0.01).4.The results observed by immunofluorescence were the same as Western Blot assays: the protein expression of ADAM10 in SH-SY5 Y cells increased under the treatment of KEN(**p < 0.01).5.Different concentrations of KEN(0-2.0 μM)had no significant effect on cell viability(p > 0.05).6.Compared with the control group(Ctrl),the secreted protein s APPαin SH-SY5Y-APP cells were significantly increased after 750 n M of KEN for 36 hours(**p < 0.01),but there was no significance in APP expression between two groups(p > 0.05).7.Compared with the control group(Ctrl),Aβ1-42 secreted by SHSY5Y-APP cells significantly decreased after 750 n M KEN treatment for36 hours(*p < 0.05).8.The ADAM10 m RNA level of SH-SY5 Y cells treated with 750 n M KEN for 36 hours had no significant change compared with the control(p >0.05).9.After treated with effective concentration of translation inhibitor reagent CHX or translation initiation factor complex competitive inhibitor reagent 4EGI-1,the induced effect of KEN on ADAM10 translation in cells could be blocked.10.After knocking down CDK5 or GSK3β,the effect of KEN on the protein expression of ADAM10 was not interrupted(**p < 0.01).11.KEN promoted ADAM10 protein expression in cells only when5’UTR fragment existed(**p < 0.01).Conclusion:1.KEN increased ADAM10 protein expression in various cells.2.The most effective condition for SH-SY5 Y cells was with 750 n M KEN for 36 hours that ADAM10 protein increased most obviously.3.The effective concentration of KEN had no effect on cell viability.4.The secreted protein s APPα in the culture medium of SH-SY5 Y cells was significantly increased after 750 n M KEN for 36 hours.5.KEN regulated ADAM10 protein expression in a 5’UTR-dependent manner at the post-transcriptional level.PART TWO: SHMT2 ACT AS AN RNA-BINDINGPROTEIN AND INVOLVED IN THE REGULATION OFADAM10 BY KENObjective:To explore the role of RNA-binding protein(RBP)during the ADAM10 translation regulated by KEN,and observe whether SHMT2 as RBP participates in the regulation of ADAM10 by KEN.Method:1.RNA pulldown assay combined with mass spectrometry analysis to detect the RNA-binding protein of ADAM10 5’UTR in SH-SY5 Y cells under the treatment of KEN compared with the control.2.The eluted 5’UTR RBPs were verified by Western Blot,and using STRING database to explore the interaction relationship of RBPs.3.The 5’UTR of ADAM10 was divided into several sequence fragments,using RNA EMSA assay to explore the binding sequence motif of 5’UTR to SHMT2.4.RNA immunoprecipitation(RIP)combined with q-PCR assays to detect the level of 5’UTR bound to SHMT2 in SH-SY5 Y cells between two groups.5.RIP-seq combined with bioinformatic method to explore the SHMT2 targeted RNA between KEN and Ctrl group,the KEGG and GO enrichment analysis described the characteristics of the common and differentially bounded RNA.6.After the SH-SY5Y-APP cells were treated with 750 n M KEN for36 hours,the differentially expressed genes(DEGs)were obtained by RNA-seq analysis.7.RNA-seq combined with bioinformatic methods to obtain the KEGG and GO enrichment analysis of DEGs.8.STRING database to explore the interaction of DEGs.9.Using Western Blot assays to detect the SHMT2 protein expression in SH-SY5 Y cells,which treated with 750 n M KEN for 36 hours.10.Using bioinformatic methods to analyze DEGs obtained by RNAseq and differentially expressed genes obtained by RIP-seq.11.Cells were transfected with si RNA of SHMT2,and Western Blot assay was used to detect whether it could block the induced effect of KEN on ADAM10 translation.Result:1.The RNA-binding protein of ADAM10 5’UTR was obtained by RNA pulldown combined with mass spectrometry analysis,part of eluted RBPs were verified by Western Blot assay,and the interaction relationship of RBPs were obtained by STRING database.2.It was observed that purified recombinant SHMT2 protein directly binds to 5’UTR in both human and mouse species by RNA EMSA assay.3.The RIP combined with q PCR assays observed that the binding of SHMT2 to 5’UTR was decreased under the treatment of KEN(**P < 0.01).4.RIP-seq combined with bioinformatic method to obtain the common and differential genes binding to SHMT2 in two groups.The gene overlapped in two groups showed that the KEGG pathways are involved in AD and neurodegenerative diseases,and play roles in the formation of methyltransferase complexes and binding function of Tau protein.Its differentially bounded genes were shown to be involved in KEGG pathways like carbon metabolism.5.RNA-seq combined with bioinformatic methods obtained differentially expressed genes(DEGs)in the transcriptome of SH-SY5 YAPP cells under KEN treatment compared with the control,and the KEGG results showed that the expression of pathways such as one-carbon metabolism was up-regulated.6.The STRING database obtained the interaction relationship of upregulated DEGs in the transcriptome,which mainly from the genes of one-carbon metabolism signaling pathway.7.SH-SY5 Y cells were exposed to a concentration gradient of KEN(0-2 μM)for 36 hours,and their SHMT2 protein expression was observed increased(*P < 0.05).8.The DEGs of RNA-seq and the differentially bounded genes of RIP-seq were analyzed together,and 112 overlapping genes were obtained,which were subjected to GO enrichment analysis according to the bioinformatic method so as to obtain the main molecular functions,biological processes,cellular component process.9.When SHMT2 was knocked down in cells,it could block the induced effect of ADAM10 translation by KEN.Conclusion:SHMT2 directly binds to the 5’UTR of ADAM10 by recognizing the GAGGG/GAAAG motif(which observed both in human and mouse species),and SHMT2 is involved in the ADAM10 translation that regulated by KEN.PART THREE: KEN ATTENUATES COGNITIVEIMPAIRMENT AND PATHOLOGICAL CHANGES INAPP/PS1 MICEObjective:To further explore the role of KEN in APP/PS1 mouse,an AD mouse model.Method:1.Take the same number of female 10-month-old APP/PS1 and littermate wild-type(WT)mice,which were divided into four groups randomly,that is,WT-saline,WT-KEN,APP/PS1-saline and APP/PS1-KEN group.By using intraperitoneal injection to treated with mouse in corresponding volume of liquid,and the volume of KEN is 5 mg/kg/2 days(KEN is fully dissolved and mixed in saline.)2.Extracting the protein of HT22 cells treated by KEN,using RNA pulldown combined with Western blot(same as the third part)assay to verify the binding of SHMT2 and 5’UTR in mice.3.After mice were treated with KEN,they were subjected to water maze and context fear conditioning tests to explore the effect of KEN on the spatial memory ability of mice.4.After the behavioral experiment of mice,the brain tissue were extracted to make brain tissue paraffin section and brain tissue lysate,respectively,and related results were detected by immunohistochemical IHC and Western Blot assays.Result:1.SHMT2 binding to ADAM10 5’UTR in HT22 cells were observed decreased by using RNA pulldown assay when treated with 750 n M of KEN for 36 hours.2.Western blot assay observed that Nr CAM and APP had no significant changes in the brain tissue of mice in each group;SHMT2 and ADAM10 were increased in the drug-added group(WT-KEN,APP/PS1-KEN)(**P < 0.01);in the APP/PS1 mouse group,the expression of s APPαin the drug treated group were increased compared with the control group(**P < 0.01);the hyperphosphorylated Tau protein were decreased in the drug-added group compared with the control(**P < 0.01).3.Immunohistochemical IHC assays explored that in APP/PS1 mice,the number of senile plaques in the APP/PS1-KEN group were significantly reduced compared with the control(APP/PS1)(**P < 0.01).4.It was observed in ELISA kit that in APP/PS1 mice,the expression of soluble and insoluble Aβ1-40/1-42 in APP/PS1-KEN group were decreased compared with the control(APP/PS1)(**P < 0.01).5.The water maze behavioral test found that in APP/PS1 mice,the escape latency of the APP/PS1-KEN group were significantly shorter than that of the control(APP/PS1)(*P < 0.05),but dwell time in quadrants(*P< 0.05)and number of platform crossings(*P < 0.05)were increased.6.Context fear conditioning tests found that,in APP/PS1 mice,the average freezing times and the average freezing time were increased(*P <0.05)in APP/PS1-KEN group compared with control.Conclusion:In vivo experiments showed that KEN could attenuate the pathological changes and spatial memory ability in APP/PS1 mouse. |
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