Mechanism Of Calcium Channel Regulated By WT1-KTS Isoform

Background:As early as the early 1990s,Wilms tumors have been found to cause abnormal kidney differentiation in areas of immature kidney tissue.In the study,it was found that there was an abnormal expression of a gene in Wilms tumors,namely Wilms Tumor 1?WT1?.WT1 was first identified as a tumor suppressor gene,which is essential for fetal development and is also involved in the development of a variety of tumors including acute myeloid leukemia.Two alternative splicing events occur on the precursor m RNA of WT1,resulting in four major WT1 isoforms.The first occurs on the fifth exon and determines the presence or absence of 17 amino acids in the protein;the second occurs on the ninth exon,making lysine-threonine-serine?KTS?insert or not into the third and fourth zinc finger structure of the protein.Due to the difference in protein structure,WT1+KTS and WT1-KTS isoforms have different affinity for DNA and RNA.WT1+KTS isoform has higher affinity for RNA,while WT1-KTS isoform has more affinity for DNA.In the previous work in the lab,it was found that there is a pyrimidine-rich region?named Tn sequence?in the ninth intron of the WT1 gene,which is composed of 32bases.This sequence can regulate the expression of WT1+KTS and WT1-KTS isoforms.When the sequence is manually deleted,the original main expression is changed from WT1+KTS to WT1-KTS.Therefore,for the purpose of subsequent research,first,at the endogenous level,CRISPR/Cas9 technology was used to precisely delete the Tn sequence from the 293T g DNA,and a stable transgenic cell line based on WT1-KTS isoform expression was successfully constructed.This provides the basis for our subsequent research.It has been reported that WT1 plays a role in regulating Ca²⁺homeostasis,but its specific mechanism is not clear.Therefore,to extend this topic,we aim to explore the molecular mechanism of WT1 subtype regulating calcium signal pathway and its function.Methods:?1?Construct over-expression WT1+KTS isoform plasmid containing Tn sequences.I use lentiviral packaging system to transfect plasmid in cells 293T-d Tn cell line?precise deletion of Tn sequences by CRISPR/Cas9 technology?to restore the expression of WT1+KTS isoform and construct a stable cell line.?2?Send RNA samples of 293T cells,293T-d Tn cells and stable cell lines of WT1+KTS isoform to do the RNA sequencing.Analyze RNA-Seq data results and perform GO analysis on differentially expressed genes.?3?Combined with the transcription factor prediction websites,screen genes whose promoter region has predicted binding sites of WT1-KTS isoform.?4?Detect expression of differential expression gene at the RNA level and protein level,and screen genes that have significant changes in expression and conform to changes subjected to RNA-Seq results.?5?Immunofluorescence technology is used to detect the localization of proteins of screened potential downstream target genes at the cellular level;?6?Use Calcium Imaging technology for next investigation.Incubating Calcium ion probe-after calcium ion influx,this metal chelate can combine with intracellular calcium ions and excite fluorescence at 494/516nm-after adding a specific activator of TRPV4,the TRPV4 channel protein is opened,then extracellular calcium ions influx into the cell,so we can observed the phenotype under different fluorescence intensity.Results:?1?After over-expressed the WT1+KTS isoform plasmid in 293T-d Tn cells,the expression changes to WT1+KTS isoform.?2?The RNA-Seq data and the GO data analysis,a large number of differentially expressed genes were found;?3?Through the transcription factor prediction websites,there are 63 genes that have binding sites with the WT1-KTS isoform.23 genes have 1 predicted binding sites,21 genes have 2 predicted binding sites,10 genes have 3 binding sites,and 9 genes have more than 3 binding sites.?4?Therefore,these 9 differentially expressed genes are targeted for the next step of detection;the expressions were detected by PCR and Western Blot.It was found that the expression of TRPV4 gene has changed and subjected to the trend of RNA-Seq data.So the next research will focus on TRPV4.?5?The results of immunofluorescence experiment showed localization is on cell membrane;?6?The fluorescence intensity shows the concentration of calcium influx into the 293T-d Tn cell is significantly weaker than that of normal 293T cells and recovering WT1+KTS isoforms cells.Conclusion:WT1-KTS isoform inhibits extracellular calcium ion influx through regulating the expression of calcium channel protein TRPV4.