Preliminary Study On The Role And Mechanism Of IGF2BP3 In Bladder Cancer

Objective: The RNA-binding protein IGF2BP3 has been reported to be abnormally expressed in a variety of tumors and affect the fate of tumor cells through m6 A regulation.In our project,we studied the role and preliminary mechanism of IGF2BP3 in bladder cancer by immunohistochemistry,cellular and molecular experiments combined with RNA sequencing and RNA methylation sequencing.Methods:(1)Screening of m6A-related RNA-binding protein IGF2BP3 by using TCGA bladder cancer gene expression data and survival data;The expression level of IGF2BP3 in tissues was detected by immunohistochemistry and qRT-PCR.(2)CRISPR / CAS9 was used to construct T24 stable cell line with IGF2BP3 knockout,DNA sequence and Western blotting were used to verify the knockout effect.The MTT test colony-forming assay,flow cytometry,scratch test and Transwell invasion test to detect changes in cell biological functions between T24 and T24 knockout IGF2BP3 cell lines.(3)We extracted and sequenced RNA from clinical bladder cancer samples and analyzed the role of IGF2BP3 in bladder cancer;At the same time,RNA-seq and MeRIP-seq were used to jointly analyze changes in cell gene expression and m6 A levels after knocking out IGF2BP3,to explore the role and preliminary mechanism of IGF2BP3 in bladder cancer.Results:(1)TCGA data analysis results suggested that IGF2BP3 is higher than normal tissues in bladder cancer(P <0.05),and patients with high expression of IGF2BP3 have shorter survival time(P<0.01);Immunohistochemical experiments showed that the positive rate of IGF2BP3 in bladder cancer tissue was 44.4%(32/72),and it was not expressed in normal bladder tissue(0/12).The difference in positive rate between the two groups was statistically significant(P<0.01);Immunohistochemistry suggested that IGF2BP3 expression was associated with lymph node metastasis and tumor infiltration(P<0.05);qRT-PCR and Western Blot experiments detected the expression of IGF2BP3 in T24,5637,J82,SW780 cells to varying degrees.(2)We successfully used CRISPR/CAS9 gene editing system to construct T24 cell line with IGF2BP3 knockout.(3)After IGF2BP3 was knocked out,the MTT experiment and the plate clone formation experiment showed that the growth and proliferation ability of T24 cells decreased;We used flow cytometry to detect increased apoptosis after knocking out IGF2BP3(P<0.05).The scratch test and Transwell invasion test showed no significant changes in cell migration and invasion ability.(4)We extracted and sequenced and analyzed RNA from bladder cancer tissues,and the results suggested that the expression changes of proliferation-related genes were related to IGF2BP3.In specimens with high expression of IGF2BP3,the expression of proliferation-related genes was also high;GO and KEGG were used to analyze T24 and T24-KO differential genes and m6A-related differential genes.The results of GO analysis suggested that differential genes were mainly enriched in extracellular matrix-related pathways,and KEGG analysis showed that differential genes were mainly enriched in TNF signaling pathway;GO analysis showed that m6 A related differential genes were mainly related to gene transcriptional regulation,and KEGG analysis of m6 A related differential genes was mainly enriched in Hippo signaling pathway;Combined analysis of differential genes at the transcription level and m6 A level suggested that the differential genes were related to cell growth and proliferation: SPHK1,POMT2,MRPS18 A.Conclusion:(1)IGF2BP3 is abnormally expressed in bladder cancer tissue;IGF2BP3 is associated with tumor metastasis and infiltration.(2)The expression of IGF2BP3 in bladder cancer tissue is related to cell proliferation genes.(3)IGF2BP3 affects the growth,proliferation and apoptosis of T24 cells.(4)SPHK1,POMT2,MRPS18 A may be the targets of IGF2BP3,but further study is needed.