Investigation Of WT1 Downstream Genes Expression Profile Change Caused By Its Endogenous Isoforms Transformation Using CRISPR/Cas9 Technology

Background: The WT1 gene is located on chromosome 11,it contains 10 exons.Two major splicing events occur on its precursor RNA,one with or without exon 5 and the other is that there are two variable 5 'splicing sites at the terminus of WT1 exon 9which is leading to the insertion or deletion of three amino acids KTS between the third and fourth zinc finger structures of WT1 protein to form either the + KTS or-KTS isoforms.WT1-KTS isoform has strong binding capacity with DNA molecules and play a role as transcription factors.And co-localize with transcription factors in cells.It has been reported in the literature that the WT1-KTS isoform can directly promote or inhibit the transcriptional level of the regulatory genes by directly binding to the promoter region of regulatory genes.Its transcriptional regulation plays a very important role in various developmental processes,including the formation of genitourinary system,retinal function and hematopoietic function.The WT1 + KTS isoforms enhanced the affinity with RNA because of the insertion of three amino acids of KTS,which greatly reduced their affinity with DNA,suggesting that they are involved in the post-transcriptional process.The main target of this study was WT1-KTS isoforms,exploring the target genes under their regulation and finding their transcriptional regulation and their effects on the biological function of cells or tumor cells.A pyrimidine-rich region,which contains 32 base in the ninth intron of WT1 gene,we named this Tn sequence.In previous experiments,we found that deletion of theTn sequence at the exogenous level resulted in a change in the 5 'splicing site that produced either isoforms of +/-KTS,leading to the conversion of the + KTS isoform-based cell line to the-KTS isoform.Thus,we have extended to the research direction of our task,namely,to verify whether deletion of Tn sequence at the genome level will also lead to the transformation of WT1+/-KTS isoforms?CRISPR/Cas9 is a technique that can precisely target nucleic acids and edit genes,so we used CRISPR/Cas9 to edit the genome of the cell,and to verify whether deletion of the Tn sequence will result in the conversion of the +KTS isoform to the-KTS isoform of WT1.If this conclusion is confirmed,we can use the obtained WT1-KTS isform cells as a raw material for the subsequent study of transcriptional regulation of the WT1-KTS isoform.Purpose:1.Using CRISPR/Cas9 technique to delete the 32-base of Tn sequence on the genome,and verify whether deletion of the Tn sequence at the endogenous level will result in the transition of the WT1 from the +KTS isoform to the-KTS isoform.2.Explore and verify downstream target genes regulated by the WT1-KTS isoform.Method:(1)To detect the expression of two endogenous WT1 +/-KTS isoforms in different cell lines and select the cell lines that are predominantly expressed the + KTS isoform.(2)The reporter gene WT1-Tn and WT1-dTn transfection respectively into the cells,to verify whether deletion of Tn sequence leads to a drastic conversion expression of-KTS isoform in the selected cell lines at the exogenous level.(3)Deletion of Tn sequences in cell lines using CRISPR Cas9 technology and verify the exogenous conclusion,screen out the cell lines which mainly expression of-KTS isoforms.(4)RNA was extracted from normal 293 T cells and 293T-dTn cells that is modified by Cas9,and RNA-seq analysis was performed to find out which genes' expression were changed,and verify by RT-PCR reaction.(5)Respectively stable overexpression the WT1 + E5 +KTS or WT1-E5 + KTS plasmids in the WT1-KTS cell line using lentivirus packaging system resulted in recorvery expression of the WT1 + KTS isoform.(6)Normal 293 T cells,Cas9 modified 293T-dTn cells and the screened stable cell lines WT1 + E5 +KTS and WT1-E5+KTS sent together to do RNA-seq analysis and screening to the genes which transcriptionnal regulated by the WT1-KTS isoform.(7)Extract RNA from the above cell lines to do RT-PCR reaction and verify that the results are consistent with the RNA-seq results.(8)Figure out the transcription factors of the target gene which is regulated by WT1-KTS.Result:(1)We selected for 293 T and TJ905 two cell lines that were mainly expressed + KTS isoforms and the ratio of +KTS isoforms in the range of 10%-20%.(2)Transfection of the reporter genes into both cell lines revealed that remove Tn sequence can increase the percentage of-KTS isoform to about 90%.(3)Using the CRISPR/Cas9 system to successfully delete the Tn sequences which leads to the proportion of WT1-KTS isoform increase to 90%,and obtain the WT1-KTS isoforms,(4)The results of RNA-seq analysis of 293 T and 293T-dTn cells found 832 genes whose expression level were changed.Among them,we selected the genes with significant difference in expression level,FBXL16 and ZNF91 these two genes are increase in their expression level when the-KTS isoform mainly increased,nine genes,ADAMTS1,LINC01021,CCND1,ESRP2,CA2,FZD10,FZD10-AS1,MMP2 and BEX1,which were decreased in their expression level after-KTS isoform increased and verified by PT-PCR reaction,found the results consistent with the results of RNA-seq.(5)WT1 + E5 + KTS and WT1-E5 + KTS cell lines obtained by lentivirusl packaging system(6)According to the RNA-seq result,nine genes up-regulated in WT1-E5 + KTS and down-regulated in 293T-dTn,thirty-three genes up-regulated in WT1 + E5 + KTS and down-regulated in 293T-dTn.Six genes down-regulated at WT1-E5 + KTS and up-regulated at 293T-dTn,twelve genes down-regulated at WT1 +E5 + KTS and up-regulated at 293T-dTn.We screened out P2RX2,TRPV4,METTL7 B,PHOSPHO1 and CTD-2162K18.4 five genes.(7)The expression of these five genes were detected by the RT-PCR reaction among the cell lines.We obtained three genes,that are TRPV4 ? METTL7 B and PHOSPHO1,whose RNA expression level is corresponds to the RNA-seq result.(8)We found three WT1-KTS binding sites in the promoter region of TRPV4,two binding sites on METTL7 B,and two binding sites onPHOSPHO1 from the transcription factor prediction website.Conclusion:1.We used the CRISPR/Cas9 technique to accurately remove the 32-nucleotide Tn sequence on the genome in the 293 T and TJ905 cell lines,and successfully verified that at the endogenous level,deleting the Tn sequence does enable WT1+KTS isoform transformed into WT1-KTS isoform.2.Via analyzing the RNA-seq result,we obtain 39 genes that exprssion are influenced by WT1-KTS,and we screen and verify the genes TRPV4,METTL7 B and PHOSPHO1 that may be regulated by the WT1-KTS isoforms.And based on the prediction of the three gene transcription factors,the promoter region of TRPV4 ?METTL7B and PHOSPHO1 both have several WT1-KTS binding sites,and we will verified the regulation in subsequent experiments and find transcription regulation mechanism.