Study On The Distribution And Function Of A New Splicing Variant Of EZH2

Alternative splicing refers to the splicing of different genes after transcription of the same gene,so that the expression of the same gene has different splicing isoforms,often corresponding to different functions.As an important component of the apparent regulatory element PRC2 complex,EZH2 is involved in the regulation of cell life activities.The splicing isoform of EZH2 has many forms in the cell.A neglected form of the splice isoform EZH2-X9 is widely expressed in SD organisms,and the specific function is still unclear.Objective: 1.Separate and identify EZH2-X9 splice isoforms,compare the nucleic acid sequence and amino acid sequence;2.Explore the expression distribution of EZH2-X9 splice isoform in cells and SD rats and follow the individual Changes in expression levels in different tissues during development;3.Preliminary investigation of the similarities and differences between EZH2-X9 splice isoforms in cells compared with ordinary full-length EZH2.Methods: 1.In PC12 cells and SD rats,the nucleic acid expression of EZH2-X9 splice isoforms was searched by PCR and gene sequencing,and the nucleic acid and amino acid sequences were analyzed by MUSCLE and BLAST;2.Fuorescence in Situ Hybridization(FISH),I mmunocytochemical technique(IF),Real-time Quantitative Polymerase Chain Reaction(q-PCR),and Western Blot(WB)to study the expression and distribution of EZH2-X9 splice isoforms in cells;in SD rats In the design and use of specific primers,q-PCR was used to study the mRNA expression distribution in different tissues,and WB was used to detect the synchrony of protein expression.3.In the undifferentiated PC12 cells,combined with genetic engineering,the fluorescence microscopy was used to observe and analyze the changes of cells,and to explore the specific functional effects of EZH2-X9 splice isoforms on NGF-induced cell differentiation.4.In PC12 cells,the gene knockdown(KD)of the homologous sequence target gene mRNA is blocked to prevent the expression of the common EZH2-WT and EZH2-X9 splice isoforms,combined with WB and protein immunoprecipitation(IP).Explore the functional differences in EZH2-WT and EZH2-X9 splice isoforms in cells.Results: 1.EZH2-X9 is widely distributed in SD rats and mainly distributed in the nucleus,in cell.2.In SD rats,the expression levels of EZH2-WT and EZH2-X9 in different tissues have certain differences,and the expression of EZH2-X9 in the brain are higher than other tissues.The growth of the individual development time is attenuated,and the expression of protein was synchronized.3.After overexpression and KD in PC12 cells,they were found to have an interaction at the mRNA level;EZH2-X9 had no significant effect on the methylation of H3K27,IP It was found that EZH2-X9 can compete with EZH2-WT in combination with EED to form PRC2 complex.4.EZH2-WT and EZH2-X9 have important functions in cell differentiation,and NGF-induced differentiation of undifferentiated PC12 cells.For KD-EZH2-X9,the total length of protrusion growth,the average length of protrusion growth,the number of protrusion growth,and the NOI% of KD-EZH2-X9 were significantly lower than those of KD-EZH2-WT;KD-EZH2-X9 would increase UIR1 and MapLC3.And the expression of GSK.Conclusion: 1.EZH2-X9 is mainly present in the brain of SD rats and is differentially expressed during development;2.EZH2-X9 and EZH2-WT compete with EED,but constitute PRC2 complex but can not mediate H3K27me3;3.EZH2-X9 and EZH2-WT regulate cell growth by regulating different target genes and molecular pathways.