Effects Of Madrasin On The Radiosensitivity Of Tumor Cells And Its Mechanism Of Action

Radiotherapy is one of the important methods of cancer treatment.Radiotherapy and radiotherapy combined with other therapies can significantly improve the survival rate of most cancer patients,but the radiation resistance of tumor tissue is still one of the reasons for the failure of radiotherapy for many solid tumors.In recent years,studies have found that alternative splicing of precursor mRNA is closely related to the occurrence and development of many tumors,and alternative splicing regulatory drugs are expected to become an effective means of cancer treatment.Madrasin is a small molecule precursor mRNA alternative splicing regulatory drug,which can interfere with the early stage of alternative splicing complex assembly,but its mechanism of action in anti-tumor and radiosensitivity is still unclear.We first conducted bioinformatics analysis of alternative splicing factors through CGGA,GEPIA2 and c Bio Portal databases to explore the abnormal changes of alternative splicing factors in tumors;then through literature research,we treated glioma U251 with alternative splicing-regulating drug madrasin with human cervical cancer HeLa cells,the regulation of madrasin on the proliferation,clone survival,migration,cycle arrest and apoptosis of U251 and HeLa cells was discussed,and the antitumor effect and molecular mechanism of madrasin in U251 and HeLa cells were studied.And further elucidated the molecular mechanism of the alternative splicing inhibitor madrasin combined with radiation to enhance the radiation sensitivity of tumor cells.Our experimental results show that:（1）splicing factors SF1,SRSF2,U2AF2,U2AF1,SF3B1,SF3A1 and RBM10 are highly expressed in gliomas,and U2AF2,U2AF1,SF3B1 and SF3A1 are related to the malignancy and prognosis of gliomas closely related;the splicing factor SRSF2 is highly expressed in cervical cancer.Subsequently,through CCK8 experiments,we found that the inhibitory effect of madrasin on tumor cells was dose-dependent in the concentration range of 0-8μM,and the median inhibitory concentrations（IC50）of U251 and HeLa cells were 2.93μM and1.68μM,respectively.Further research found that madrasin induced G1 arrest in U251and HeLa cells,and down-regulated the cycle-related proteins CDK2,Cyclin E1 and other proteins.In addition,madrasin,an alternative splicing inhibitor,inhibited the splicing of the precursor mRNAs of apoptotic genes Tp73 and bc L-x in a dose-dependent manner,resulting in two splicing isoforms produced by Tp73 and bc L-x Expression imbalance,which in turn promotes tumor cell apoptosis.This study revealed that madrasin,as an inhibitor of pre-mRNA splicing regulation,may be a potential drug for the treatment of glioma and cervical cancer.（2）Firstly,HeLa cells were irradiated with heavy ions.The sequencing data of alternative splicing of precursor mRNA showed that:2 Gy and 4 Gy heavy ion irradiated HeLa cells had a higher proportion of exon skipping and intron retention;2Gy heavy ion irradiated HeLa cells can activate damage repair-related signaling pathways,and 4 Gy heavy ion irradiation of HeLa cells can activate p53,autophagy and apoptosis-related signaling pathways.Further using low-concentration madrasin combined with radiation,the results showed that 0.25μM madrasin combined with 4Gy X-ray or 2 Gy ¹²C⁶⁺effectively inhibited the proliferation and survival of U251 and HeLa cells;induced U251 and HeLa cells G1 phase arrest;down-regulated cycle-related Protein CDK2,Cyclin E1 and other proteins.In addition,madrasin induced up-regulation of TAp73 expression and down-regulation ofΔNp73 expression,activating the caspase-mediated mitochondrial apoptosis pathway.The above results indicate that low concentrations of madrasin combined with radiation may regulate the radiosensitivity of tumor cells through TAp73/ΔNp73.The results provide a theoretical reference for the application of madrasin as a radiosensitizing drug in clinical treatment.