The Function And Regulation Of Transcription Factor Ikaros In Lung Cancer Progression

Objective:Firstly, we try to learn the function of Ikaros in lung cancer development, to detect the influence of Ikaros expression on lung cancer cell behavior, and form the link between Ikaros expression in lung cancer tissue and clinical lung cancer stages. classification, and prognosis, we try to determine the ectopic expression mechanism of lung cancer cell through the study of the methylation. histone modification, and chromosome higher structure. Last we hope to enrich the knowledge of lung cancer initiation, and provide data for the clinical diagnosis and treatment of lung cancer.Methods:1. Function study of Ikaros in lung cancer progressionExpression level of Ikaros are detected by Western blot in several solid tumor cell lines, including:H209and H69. which are the members of small cell lung cancer: H1155. A549. H441. LLC1. which belong to none small lung cancer cell; MCF-7. MDA-MB-231. and SKBR3, which are breast cancer cells:HUVEC(HV). Beas2B. which are normal epithelial and endothelial cell; The loss-of-function and gain-of-function assay were used to investigate the function of Ikaros. Wound healing and Transwell test were performed to detect the effect of Ikaros on behavior. RT PCR was used to detect the expression level of the genes involved in cell invasion and migration. Immunohistochemistry was used to test the Ikaros expression level in clinical human lung cancer samples.2. The epigenetic regulation of Ikaros ectopic expression in lung cancer cellsThe IKZF1conserved sequences between human and mice genomes were searched using BLAST software. Bisulfite-Sequencing method was used to detect the DNA methylation status of IKZF1promoter in U937. H1155cell lines and so on. The distribution of H3K27ace. H3K4monome, H3K9ace. H3K4me2and H3K4me3on Ikaros gene was detected by ChIP.3C assay was used to investigate the regulation mechanism of IKZF1expression in human lung cancer on chromosome level.Results:1. Ikaros inhibits lung cancer progression Western blot demonstrated H209. H69. H1155,H441and SKBR3cells express Ikaros. H69cells changed cell shape from floating to attachment after Ikaros knockdown. A549overexpressing Ikaros lost attachment to the culture plate. In A549overexpressing Ikaros cells the migration and invasion were reduced. It was found that Ikaros inhibited the transcription of the genes such as Nanog, ErbB2. SIP1, TGFa and a-SMA. Finally, immunohistochemistry demonstrated that low differentiated lung cancer tissues express more abundant Ikaros than the higher differentiated ones.2. DNA demethylation is the course of Ikaros ectopic expression in lung cancerBLAST software is used to compare the IKZF1between human and mice, homo1. homo2……homoll are found to be homologous. ChIP results demonstrate that H3K27ace and H3K4monome enrich in homo4. homo5. homoll in U937. in HI155. they don’t, these results demonstrate that homo4. homo5. and homoll are potential enhancers.3C results demonstrate that in spatial structure, promoter approach to enhancers in U937. not in H1155. which further substrate in H1155enhancers don’t work. H3K9ace. H3K4me2. me3enrich in the transcription start sites of U937. not in H1155. Bisulfite-Sequencing demonstrate that DNA methylation degree is significantly lower in U937. H1155than the cells which don’t express Ikaros. meaning that DNA demethylation is the expression reason of Ikaros H1155.Conclusion:In lung cancer cells, the ectopic expression of Ikraos suppresses the migration and invasion through inhibition of the transcription of Nanog. ErbB2. SIP1. TGFa and a-SMA. It is consistent with the report that Ikaros is a suppressor of ALL. Based on our findings, it is suggested that ectopic expression of Ikaros in lung tumors contribute to tumor cell differentiation and inhibit tumor progression consequently.In lymphocytes IKZF1is regulated by several enhancers which interact with promoter directly and form loop structure to active IKZF1transcription. However, in lung cancer cells, the ectopic expression of Ikaros is activated by the DNA demethylation but not the enhancers in IKZF1promoter.