Preliminary Study For Mechanism Of TNIK Gene Affecting The Antipsychotic Efficacy

Object:Traf2-and NCK-interacting kinase(TNIK)gene is an important schizophrenia susceptibility gene and is also involved in response to antipsychotic drugs,but the mechanism of TNIK gene affecting the efficacy of antipsychotics is unknown.In this study,three SNPs of TNIK(rs2088885,rs7627954 and rs13065441)were selected to explore the relationship between TNIK gene polymorphism and the risperidonen response.At the same time,the TNIK differentially expressed cell models were constructed and intervented by antipsychotic drugs.The expression levels of the related genes and molecular proteins before and after the treatment in each cell model were compared to explore the mechanism by which the TNIK gene affects the efficacy of antipsychotic drugs.Method:1.Recruiting 148 Chinese Han schizophrenia patients who have never been treated with drugs or whose drug cleaning period has been longer than five metabolic halflife periods.The enrolled subjects have been prescribed single risperidone during the study.Clinical effect was assessed on the Positive and Negative Syndrome Scale(PANSS)and SNP genotyping was undertaken using the Mass ARRAY? SNP IPLEX platform(Agena Bioscience?,San Diego,CA,USA).2.Transfecting the U251 cells with TNIK plasmid,empty plasmid and TNIK Si RNA by Lipofectamine to construct the TNIK gene overexpression cell model,the control cell model and the TNIK gene knokdown cell model.Then these cell models were treated with clozapine and risperidone at 50% concentration of inhibition for 24 h.Western-blot was used to detect the proteins expression levels of Wnt / β-catenin pathway in various cell models before and after drug intervention,including GSK-3β,phosphorylated GSK-3β,β-catenin and TCF4.3.Transcriptome sequencing was performed on the risperidone intervention groups and blank groups of the TNIK differentially expressed cell models,and the GO enrichment analysis was performed on the differentially expressed genes Result:1.Single loci analysis:The allele(χ~2 = 5.257 p = 0.022)and genotype(χ~2 = 6.647 p =0.036)frequencies of TNIK rs2088885 had significant associations with the efficacy of risperidone;the allele frequency of TNIK rs7627954 had significant associations with the efficacy of risperidone(χ~2 = 4.472 p = 0.034).High linkage disequilibrium was observed between SNPs rs2088885 and rs7627954,so we assess the association between the haplotypes and treatment responses.The result revealed that the haplotype CT was more prevalent in good responders than in poor responders(p=0.0451).Ggenotypes of rs2088885 and rs7627954 were related to the efficacy of risperidone under co-dominant,recessive,and additive genetic model,and survive the Bonferroni correction(p<0.05);The association between TNIK rs13065441 and risperidone responses was not found in this study.2.The interventions of clozapine and risperidone in U251 cells can significantly reduce the expression levels of phosphorylated GSK-3β,β-catenin and TCF4.The expression level of GSK3βdecreased in the clozapine intervention group(p=0.025),but not in the risperidone intervention group(p >0.05).The results of Western blot indicated that the activity of Wnt pathway was inhabited by risperidone and clozapine;3.There were no statistically significant differences in expression levels of GSK-3β,phosphorylated GSK-3β,β-catenin between TNIK overerpression grop and control grop or between TNIK knockdown group and control group,but TCF4 expression levels was significantly higher in TNIK knockdown group.It is suggested that TNIK gene has no significant effect on the activity of Wnt pathway,but the inhibition of TNIK expression will increase the expression of TCF4,a downstream molecule of Wnt pathway.4.Under the clozapine intervention,the expression levels of GSK-3β,phosphorylated GSK-3β and β-catenin did not change significantly in the TNIK overexpression group and the TNIK knockdown group from those in the control group,but TCF4 expression levels was significantly higher in TNIK knockdown group(p=0.028).It is indicated that the down-regulation of Wnt pathway by clozapine was not affected by TNIK expression levels.5.Under the risperidone intervention,there was no significant differences in the expression levels of GSK3,β-catenin and TCF4 between the TNIK overexpression group and the control group(p > 0.05),and the expression level of phosphorylated GSK3 was lower in the overexpression group(p = 0.021).The expression levels of phosphorylated GSK3,β-catenin and TCF4 was significantly higher in TNIK knockdown group than that in the control group(p = 0.031)under the risperidone intervention,which indicated that knockdown of TNIK gene could reverse the down-regulation effect of risperidone on Wnt / β-catenin pathway to a certain extent.6.The result of whole transcriptome resequencing:Genes expressed differentially between the TNIK knockdown group and the control group were enriched in pathways related to axonogensis,glial cell differentiation,and neuron migration;the expression levels of many genes changed after the risperidone intervention,and these genes were enriched in the pathways related to cognition(GO:0050890)in TNIK knockdown group but not in control group or TNIK overexpression group.Conclusion:1.Our findings sugesst that TNIK genes rs2088885 and rs7627954 are related to the efficacy of risperidone.2.Risperidone and clozapine could decrease Wnt / β-catenin pathway activity,and knockdown of TNIK gene could reverse the down-regulation effect of risperidone on Wnt / β-catenin pathway to a certain extent.3.Transcriptome sequencing results suggest that the expression levels of many genes changed after the risperidone intervention,and these genes were enriched in the pathways related to cognition(GO:0050890)in TNIK knockdown group but not in control group or TNIK overexpression group.