Alterations Of BTG1and Its Regulatory Pathway After Heavy Ion Irradiation

The The question of how to improve the radiosensitivity of tumor cells duringheavy ion cancer therapy has always been a critical problem in the field ofradiobiology. The alterations of radiation-related proteins and their regulatorypathways provide potential therapeutic strategies. Therefore, investigation of the keyfactors and regulatory pathways of these proteins in response to heavy ion radiationwill provide basic experimental data and practical applications to enhance theradiosensitivity of tumor cells.B cell translocation gene1(BTG1) is a member of BTG/TOB family, which has acommon highly homologous domain and plays a key role in the regulation of cellproliferation and cell cycle progression. It is widely reported that other members ofthis family are involved in cellular radiation response. However, the role of BTG1inradiation response is still unclear and whether BTG1could be served as a target forsensitizing tumor cells to ionizing radiation (IR) needs to be elucidated. BTG1isrecognized as a strong anti-proliferative gene to regulate cell growth. Expression ofBTG1not only inhibits the proliferation of cells but also plays important roles invarious other physiological progresses such as cell cycle, apoptosis and differentiation.Therefore, BTG1might be involved in the more general processes of cell cycle controland in cellular responses to stress, it is necessary to focus on the specific role of BTG1in response to heavy ion radiation. In this study, the human renal carcinoma786-O cells were treated with heavy ionor X-ray irradiation. To verify that BTG1is induced in response to IR, the expressionof BTG1was analyzed by quantitative polymerase chain reaction and western blotting.By bioinformatics and dual luciferase reporter experiments, two microRNAs werevalidated to target BTG1. The influence of alterations of BTG1protein levels on cellsurvival, apoptosis, genomic instability and radiosensitivity of786-O cells wasexplored. The main results of the study were summarized as follows:1. The induction of BTG1occurred rapidly at the protein and mRNA levels after5Gy of X-rays or2Gy of carbon ion beam irradiation.2. Overexpression of BTG1by transient transfection of BTG1vector into786-Ocells promoted cell survival, while decreased apoptosis and genomic instabilityinduced by IR. Conversely, knockdown of BTG1decreased cell viability, induced cellapoptosis and genomic instability, therefore enhanced the cellular radiosensitivity.3. Both miR-454-3p and miR-19b target BTG1mRNA. The negative correlationbetween the expression of these miRNAs and BTG1was observed in786-O cellsexposed to IR. Suppression of BTG1by miR-454-3p led to inhibition of cell survivaland enhancement of cell radiosensitivity.4. The down-regulation of BTG1was considered to closely associate with theprocess of S phase through detection of the BTG1mRNA levels in different cell cyclephases in MRC-5cells. Down-regulation of BTG1will prevent the activation of theG1checkpoint and lead to damage accumulation during S phase, resulting in S phasearrest.5. An interpretation of NF-κB signal transduction pathway to promote cellapoptosis in the presence of IR was proposed through determination of NF-κB, anupstream regulatory factor of BTG1, and PRMT1, a methyltransferase whose activitywas dependent on BTG1amount.In conclusion, our results revealed the alterations of BTG1and its regulatory pathway induced by heavy ion irradiation. This is the first report on BTG1as aresponder to IR. BTG1is a direct target of miR-454-3p and miR-19b and can be usedas a potential therapeutic target to sensitize tumor cells to IR. The classic NF-κB signalpathway can be complemented through regulation of BTG1to promote cell apoptosis.This research provides a potential method to enhance tumor cellular radiosensitivity inheavy ion radiotherapy.