The Roles And Mechanism Of PRMT5S In Radiationinduced Cell Death

Radiotherapy is a routine treatment for malignant tumors,which can cause different degrees of autophagy and apoptosis in various tumor cells such as breast cancer,prostate cancer,lung cancer and colorectal cancer,and thus have a significant effect on the therapeutic effect of tumors.Apoptosis,a type I programmed cell death,removes potentially dangerous cells that are not conducive to the body.It is an active process that maintains the homeostasis of the body.The outcome of autophagy is different from that of apoptosis,which can cause adaptive response and survival,as well as autophagic cell death,depending on different cell types,intensity of stimulation,and environmental factors.The relationship between autophagy and apoptosis,either independent or interaction and molecular transformation,understanding the mechanism of cell death is of great value for clinical radiotherapy of tumors.Protein arginine methyltransferase 5(PRMT5)plays an important role in arginine methylation of proteins,is involved in the regulation of the signaling pathways of various signaling molecules in cells,and is involved in the regulation of various tumor-associated proteins,affecting cells.Various life processes such as proliferation,differentiation and apoptosis.A number of studies have shown that PRMT5 is highly expressed in a variety of tumors and is closely related to tumor formation and development as well as prognosis.PRMT5 has several different selective shears,and PRMT5 S is one of them.Previous studies have found that ionizing radiation can induce cells to express PRMT5 S,and it can exert radiation sensitization.This study focuses on the role of PRMT5 S in ionizing radiation-induced cell death and its related mechanisms.Objective:Human cervical cancer HeLa cells were used as the research object to investigate the role of PRMT5 S in ionizing radiation-induced cell death and its possible regulatory mechanisms,which provided a new basis for clinical gene therapy of tumors.Methods:(1)Cell line: human cervical cancer HeLa cells;(2)Irradiation conditions:X-RAD 320 iX biological irradiation instrument,voltage 180 kV,current 20 mA,absorbed dose rate is 1.0Gy / min;(3)Using calcium phosphate co-precipitation transfection method to construct PRMT5 gene Silencing the cell model,and then constructing different PRMT5 phenotype cells(MYC,PRMT5 L,PRMT5S)by liposome transient transfection;(4)Using CCK8 to detect survival rate;(5)Using colony formation assay to detect cell radiation sensitivity;(6)RT-PCR and Western Blot were used to detect changes in mRNA and protein levels;(7)Flow cytometry was used to detect autophagy rate and apoptosis rate.Results:1.IR induced HeLa cell death and expression of selective splicing PRMT5SThe HeLa cells were treated with ionizing radiation.The CCK8 experiment showed that the cell viability of the 8Gy group was significantly lower than that of the sham group,and the trend of the survival rate decreased in a time-dependent manner,indicating that ionizing radiation can induce cell death.Western Blot showed that compared with the sham group,the expression of autophagy-related protein LC3-II was significantly increased in the 8Gy group,and the expression of the apoptosis-related protein Cleaved Caspase-3 was significantly increased.The selective cleavage PRMT5 S protein The level of expression is significantly increased.Flow cytometry experiments showed that compared with the fake irradiation group,the autophagy rate and apoptotic rate of the 8Gy group were significantly increased(P<0.05).These results suggest that ionizing radiation can induce autophagy and apoptosis in cells,and can increase the expression of PRMT5 S protein.2.The roles of PRMT5 S in the radiation-induced cell deathThe PRMT5 silencing model was treated with ionizing radiation.The colony formation experiment showed that compared with the empty vector Control group,after the PRMT5 was silenced,the clonality of the cells gradually decreased with the increase of the radiation dose.The CCK8 experiment found that compared with the fake irradiation group,the cell viability of the 8Gy group was significantly reduced in the gene silencing group.The above results suggest that silencing PRMT5 cansignificantly increase radiation-induced cell death and increase cell radiation sensitivity.After 8Gy irradiation,compared with the sham group,Western Blot showed that the expression level of autophagy-related protein LC3-II was significantly decreased in the gene silencing group;the flow cytometry experiment also found that the gene silencing group ShPRMT5 The phagocytosis rate(14.07%)was significantly lower than that of the empty vector Control group(23.96%).The above results suggest that silencing PRMT5 can significantly reduce the level of radiation-induced autophagy.After 8Gy irradiation,Western Blot showed that the expression level of apoptosis-related protein Cleaved Caspase-3 was significantly increased in the gene silencing group compared with the sham group.The results of flow cytometry also showed that the 8Gy group cells The apoptotic rate was significantly increased in the gene silencing group(11.37%).The above results suggest that silencing PRMT5 can significantly increase the level of radiation-induced apoptosis.The cells treated with different PRMT5 phenotypes(MYC,PRMT5 L,PRMT5S)were treated with ionizing radiation.The CCK8 experiment showed that the cell viability of the 8Gy group was lower in the overexpressed PRMT5 S group(33.96%)than the sham group.Overexpression of PRMT5 S significantly increased radiation-induced cell death;after the addition of the autophagy inhibitor 3-MA and the apoptosis inhibitor ZVAD-FMK,the CCK8 experiment found that after 8 Gy irradiation,the three cells were compared to the sham group.The survival rate was reduced,and the most significant decrease was observed in the overexpressed PRMT5 S group,and the pro-death effect of PRMT5 S was confirmed once again.Compared with the untreated Control group,the survival rate of the three cells decreased in the 3-MA group(P<0.05),and increased in the ZVAD-FMK group(P<0.05),including the PRMT5 S group.The change is most obvious.The above results suggest that inhibition of autophagy can promote death,and autophagy has a protective effect here.Western Blot showed that compared with the sham group,after 8Gy irradiation,the expression of autophagy-related protein LC3-II was significantly decreased in the overexpressed PRMT5 S group,and the expression of apoptosis-related protein Cleaved Caspase-3 was significantly increased.Expression of PRMT5 S significantly inhibited radiation-induced autophagy and promoted apoptosis induced by radiation;after adding autophagy inhibitor 3-MA andapoptosis inhibitor ZVAD-FMK,Western Blot experiment found that with unmedicated Control Compared with the cells,after 8Gy irradiation,the expression level of apoptosis-related protein Cleaved Caspase-3 was decreased in the ZVAD-FMK group,and increased in the 3-MA group,especially in the overexpressed PRMT5 S group.The highest results suggest that inhibition of radiation-induced autophagy can promote apoptosis,and PRMT5 S affects the transition from autophagy to apoptosis.3.The effect of PRMT5 S on histone after ionizing radiationThe PRMT5 gene silencing model was treated with ionizing radiation.Western Blot experiments showed that the expression levels of arginine methylated protein of H4R3 and H3R2 in ShPRMT5 cells were significantly lower than those in the empty vector Control group after 8Gy irradiation.Ionizing radiation treatment was performed on cells with different phenotypes of PRMT5(MYC,PRMT5 L,PRMT5S).Western Blot experiments showed that the level of histone methylation in the cell model of PRMT5 changed after 8 Gy irradiation compared with the sham group.Not obvious.H1 C overexpression plasmid was co-transfected into cells of different PRMT5 phenotypes and then treated with ionizing radiation.Western Blot assay showed that autophagy-related protein LC3-II was co-transfected with H1 C compared with transfected PRMT5 S alone.The expression level was significantly increased.The above results suggest that PRMT5 S can affect the autophagy induced by radiation by histone H1 C,and thus participate in the regulation of cell death.Conclusions:1.Ionizing radiation can increase the expression of the selective splicing PRMT5 S,and PRMT5 S can increase radiation-induced cell death,thereby achieving radiation sensitization.2.PRMT5 S can inhibit radiation-induced autophagy,promote radiation-induced apoptosis,and affect the transition from autophagy to apoptosis.3.PRMT5 S affects the autophagy and apoptosis induced by radiation by histone H1C,and then participates in the regulation of cell death.