The Effect And Mechanism Of Pokemon Regulating IGFBP3 On The Metastasis Of Bladder Cancer

BackgroundIn our country,urothelial carcinoma(UC)is still the most common urinary system malignant tumor.However,to date,compared with cytotoxic therapies,no clinical trials for UC targeted drugs have proven to improve the survival rate.The Cancer Genome Atlas(TCGA)has detected many potentially important genetic changes in bladder cancer,providing a roadmap for the use of small molecule inhibitors in this disease.In addition,in the past five years,with the rapid development of next-generation sequencing technology,deep sequencing has defined the molecular characteristics of tumors in real time.Finding specific targeted genes for the treatment and diagnosis of bladder cancer has become an urgent problem in the era of precision treatment.ObjectiveFigured out the expression of Pokemon in bladder cancer and combined in vivo and in vitro experiments to clarify that Pokemon is related to the malignant behavior of bladder cancer in vivo and in vitro.The "rescue experiment" was used to clarify that Pokemon can regulate the target gene IGFBP3,and to further clarify that IGFBP3 inhibits the malignant biological function of bladder cancer.The application of high-throughput chip analysis and the use of bisulfite modified genome sequencing revealed that Pokemon exerts the molecular mechanism of inhibiting bladder cancer metastasis by influencing IGFBP3 epigenetic modification,and actively seeks downstream pathways regulated by IGFBP3.Furthermore,it provides theoretical basis and clinical data support for actively looking for new methods for targeted treatment of bladder cancer.Methods1 Determine that Pokemon is highly expressed in bladder cancer and plays a role in promoting cancer(1)Perform immunohistochemical staining,western blot,and q RT-PCR on clinical tissue samples to detect the expression of Pokemon in the tissue(2)Detect the expression of Pokemon in bladder cancer cells and bladder epithelial immortalized cells.(3)Construct bladder cancer cells that overexpress/knock down Pokemon by overexpression plasmid(pcDNA3.1)and small interfering RNA(siRNA)(4)Through cell-cell adhesion test,cell matrix adhesion test,Transwell cell invasion and migration test,anoikis test,analyze the role of Pokemon in these tumor metastasis phenotypes.Finally,the mRNA and protein expression levels of related functional genes,Integrin-?1,MMP-9,and BCL-2 were detected by q RT-PCR and western blot.2.Determine the expression of IGFBP3 in bladder cancer and its anti-tumor effect(1)Perform immunohistochemical staining,western blot,and q RT-PCR to detect the expression of IGFBP3 in the tissues from clinical tissue samples(2)Detect the expression of IGFBP3 in bladder cancer cells and normal bladder epithelial immortalized cells.(3)Construct overexpression/knockdown IGFBP3 cells by overexpression plasmid(pcDNA3.1)and small interfering RNA(siRNA)(4)To analyze the role of IGFBP3 in the above-mentioned tumor metastasis phenotype.Detect the mRNA and protein expression levels of related functional genes by q RT-PCR and western blot3.Prove that Pokemon affects the metastasis of bladder cancer through targeted regulation of IGFBP3Design a rescue experiment,knock down Pokemon and IGFBP3 at the same time,and judge whether the tumor metastasis phenotype and related functional protein expression are restored compared with knocking down Pokemon alone4.Verify the epigenetic mechanism of Pokemon regulating IGFBP3,and find the downstream signaling pathway of IGFBP3.(1)Transfect siPokemon and FAM nonsense sequences in bladder cancer cells,and then perform genome sequencing after bisulfite modification.According to the sequencing results,the specific CpG sites where methylation occurred in the promoter region of IGFBP3 were analyzed,and the methylation map was drawn.Analyze the degree of methylation of the IGFBP3 promoter in the negative control group and the Pokemon knockout group.(2)Transfect the pcDNA3.1 empty vector and pcDNA3.1IGFBP3 into bladder cancer cells.After culturing for 24 hours,collect the cells and perform transcriptome sequencing.Statistical analysis lists all up-regulated/down-regulated genes that are statistically different after overexpression of IGFBP3.Screen the signal pathways related to tumor metastasis from these genes.After determining the signal pathway,perform real-time q RT-PCR and western blot to verify the signal pathway.5.Establishment of animal model for in vivo experimentsT24 cells stably knocked out/overexpressing Pokemon and IGFBP3 were injected into the tail vein of nude mice to establish a tail vein metastasis model of bladder cancer in nude mice.The mice were sacrificed 8 weeks later,the tumor metastasis was detected by laparotomy,and the number of metastatic tumors was counted visually.And HE staining the tissue sections to determine tumor metastasis.Results1.The high expression rate of Pokemon in bladder cancer tissues and adjacent tissues is significantly different.The high expression rate of cancer tissue is 67.3%,and the high expression rate of adjacent tissues is 26.9%.The mRNA and protein expression of Pokemon in bladder cancer cell lines are higher than normal epithelial immortalized cells.Analysis of clinical specimen information showed that Pokemon expression is significantly correlated with the tumor stage of bladder cancer and distant metastasis,but the correlation with age,gender,and tumor size is not statistically significant.2.Pokemon can promote the invasion,migration and cell matrix adhesion of bladder cancer cells in vitro;Pokemon can inhibit cell-cell adhesion and anoikis;Pokemon can promote the transcriptional expression of MMP-9,Integrin-?1,and BCL-2.3.There is a significant difference in the low expression rate of IGFBP3 in bladder cancer tissues and adjacent tissues.The high expression rate of cancer tissue is 63.5%,and the low expression rate of adjacent tissues is 38.5%.The mRNA and protein expression of IGFBP3 in bladder cancer cell lines are lower than those of normal bladder epithelial immortalized cells.Analysis of clinical specimen information showed that the expression of IGFBP3 was significantly correlated with distant metastasis of bladder cancer,but there was no statistically significant correlation with age,sex,tumor size,and tumor stage.4.IGFBP3 can inhibit the invasion,migration and cell matrix adhesion of bladder cancer cells in vitro;IGFBP3 can promote cell-cell adhesion and anoikis;IGFBP3 can inhibit the transcriptional expression of MMP-9,Integrin-?1,and BCL-2;IGFBP3 can Inhibit the co-localization of VASP and Paxillin,which represents the inhibitory effect of IGFBP3 on focal adhesion formation.5.Pokemon can regulate the transcription and expression of IGFBP3,promote the invasion and migration of bladder cancer,cell-matrix adhesion,and inhibit cell-cell adhesion and anoikis of bladder cancer.Pokemon can promote the transcriptional expression of MMP-9,Integrin-?1 and BCL-2 by regulating the transcriptional expression of IGFBP3.6.Compared with the negative control group,the siPokemon group that knocked down Pokemon in T24 cells had a decrease in the methylation ratio of 16 sites and an increase in the methylation rate of 2 sites.Compared with the negative control group,the ratio of methylation at 10 sites in the siPokemon group of J82 cells decreased and the ratio of methylation at 3 sites increased.The percentage of the total methylation sites of 38 sites showed that compared with the control group,the percentage of IGFBP3 promoter methylation sites in the T24 and J82 cells of the siPokemon group decreased,which proved that when Pokemon expression decreased,it would make IGFBP3 promoter is demethylated to restore the transcriptional expression of IGFBP3.7.Eukaryotic transcriptome sequencing found that the overexpression of IGFBP3 can increase the expression of RIG-1,p<0.05.The results of verification experiments on the signaling pathway showed that after knocking down Pokemon or overexpressing IGFBP3,the mRNA and protein expression levels of RIG-1 increased,while after overexpressing Pokemon or knocking down IGFBP3,the mRNA and protein expression levels of RIG-1 are reduced.The "rescue" experiment results showed that when siPokemon and si IGFBP3 were co-transfected,the transcriptional expression level of RIG-1 was significantly lower than when Pokemon was knocked down alone.8.In in vivo animal model experiments,a large number of metastatic tumor nodules were found in the lungs of mice in the negative control group,and the number of metastatic nodules in the lungs of mice in the knockdown Pokemon group and mice overexpressing IGFBP3 was significantly reduced compared with the control and the size is significantly smaller.The results of HE staining were consistent with tissue performance.The above proves that the knockdown Pokemon and over-expression IGFBP3 group nude mice showed similar metastasis inhibition characteristics.ConclusionPokemon can regulate the transcriptional expression of IGFBP3 by affecting the degree of methylation of the IGFBP3 promoter,thereby promoting the metastasis of bladder cancer.Moreover,Pokemon-IGFBP3 in bladder cancer can regulate the downstream RIG-1 signaling pathway.