The Mechanism Studies On Epigenetic Modification Participating In Radiation Response In Carcinoma Cells

Epigenetic modulation plays important roles in many cellular processes including tumorigenesis,autoimmune disease,cell differentiation and development.It includes an array of molecular modifications such as DNA methylation,histone modifications,micro RNAs and so on.In current research,to understand the new mechanism of radiation induced biological effects and to provide theoretical basis and new targets for cancer radiotherapy combined the epigenetic modulation,we investigated the radiation response involving the epigenetic modulations and their mechanisms in the two-dimensional(2D)cells or three-dimensional(3D)cultured carcinoma cells.Using the techniques of karyotype analysis and gene interference in 2D cultured carcinoma cells,we explored the correlation between micro RNA expression and the phenomenon of radiation induced premature chromatid separation.Through the gene expression sequencing,Ch IP-seq and Pull-down assay,we investigated the effect and the signal pathway of the histone lysine methyltransferases SET and H4K20 methylation participating in the radiation response in cells.We inspected the key factors which induce the radioresistance of 3D cultured carcinoma cells via the PCR array,MSP and gene interference.We found that:(1)Mi R-142-3p responded to radiation and participated in the radiation induced premature chromatid separation.The expression of Bod1(Biorientation of chromosomes in cell division 1),a novel kinetochore protein required for chromosome biorientation,is regulated by mi R-142-3p through targeting the 3’-UTR of Bod1 gene.Overexpression of mi R-142-3p increased the premature chromatid separation and G2/M cell cycle arrest by suppressing Bod1 expression.Either overexpression of mi R-142-3p or knockdown of Bod1 sensitized carcinoma cells to X-ray radiation.Overexpression of Bod1 inhibited radiation and mi R-142-3p induced premature chromatid separation and increased resistance to radiation in carcinoma cells.(2)SET8 responded to the radiation and suppressed the monomethylation of H4K20(H4K20me1)in the irradiated cells.Knockdown of the expression of SET8 suppressed the accumulation of 53BP1 in the site of DNA damage and increased the radiosensitivity in carcinoma cells.The primary results suggest that multiple genes such as RNF8,BARD1,PAK6 and CDK10 were involved in the progress of radiation induced DNA damage repair regulated by SET8 through the gene expression sequencing,Ch IP-seq and Pull-down assay.(3)The expressions of some cell cycle regulation genes in 3D cells were lower compared with those in the 2D cells and the promotors of these genes were hypermethylated in 3D A549 cells.The treatments of irradiation or 5-Aza-Cd R induced the demethylation in the promotors of above genes and resulted in the increased expressions only in 3D cells.The further research showed that the low expression of RBL1 owing to the hypermethylation of the promotor in 3D cultured cells enhanced the radioresistance and overexpression of RBL1 sensitized the carcinoma cells to irradiation.Our findings indicate that three main epigenetic modifications participate in the response to irradiation in cells and affects the radiosensitivity of cells,which provides the new targets for cancer radiotherapy combined the epigenetic modulation.The 3D culture in vitro is a new cell culture model that more closely mimics the physiology features,which sheds a new light on medcine development and improves the understanding about the radiation induced biological effects.