| CRC is the third most common cancer in the world,as well as the fourth most deadly cancer and has the fifth highest mortality rate of all cancers,and its incidence is increasing year on year.and the main treatment is surgical treatment adjuvant radiotherapy.However,the radiation resistance of tumor cells remains the biggest obstacle limiting the effect of radiation therapy for various human tumors,including colon cancer,leading to increased tumor recurrence and reduced treatment effect,and it is of great clinical significance to find various other auxiliary means to improve their radiation sensitivity.Arginine methylation is a recently discovered post-transcriptional modification catalyzed by arginine methyltrans ferases(PRMTs).Protein arginine methylation is widely present in organisms and plays many important roles,such as involvement in post-transcriptional regulation,RNA processing,cell proliferation,apoptosis,differentiation,and tumor formation,and proteins with abnormal methylation levels can be used as potential biomarkers or drug research targets.At present,there are 11 kinds of PRMT reported,of which type Ⅰ(PRMT1,PRMT2,PRMT3,PRMT4,PRMT6 and PRMT8)catalyzes monomethyⅠ and asymmetric dimethyⅠ,type Ⅱ(PRMT5,PRMT9)catalyzes symmetric dimethyⅠ,and type Ⅲ(PRMT7)can only catalyze monomethyl.CARM1,also known as PRMT4,is a member of the PRMT family,belongs to type Ⅰarginine methylase,its expression level is related to the prognosis of cancer has been shown to be overexpressed in various human cancer types,as a transcription factor involved in the apoptosis,invasion and metastasis of tumor cells.In this study,HCT116 and HT29 cells were used as the main subjects to explore the role of CARM1-mediated arginine methylation in radiation sensitivity and its possible related mechanisms.Objective:In this study,the radiation sensitization of CARM1-mediated arginine methylation in colon cancer cells HCT116 and HT29 cells and its possible mechanisms were investigated.Methods:1.X-RAD 320i X deep irradiator is used for irradiation.The irradiation conditions:voltage 180 kV,current 20 mA,target distance 70 cm,dose rate 1.0 Gy/min and voltage 320 kV,current 16.5 mA,target distance60cm,dose rate2.22 Gy/min.2.Using a bioinformatics approach to analyse CARM1 in colon cancer tissues at different stages and their impact on survival in patients with COAD3.CCk-8 method:By detecting the effect of CARM1 inhibitor SGC2085 on the proliferative activity of HCT116 and HT29 cells,the optimal drug concentration for radiation sensitization experiment was obtained.The inhibitory effect of different irradiation doses on HCT116 and HT29 cells was detected to obtain the optimal irradiation dose.To detect the proliferation viability of the SGC2085 on HCT116 and HT29 cells before and after irradiation4.Clone formation experiment:To detect the effect of CARM1 inhibitor SGC2085 combined with ionizing radiation on the colony formation capacity of HCT116 and HT29 cells.5.Wound healing experiment and Transwell method:the effect of CARM1 inhibitor SGC2085 combined with ionizing radiation on the migration and invasion capacity of HCT116 and HT29 cells6.Flow cytometry:To detect changes in apoptosis,cycle,autophagy and ROS of HCT116 and HT29 cells by the combination of CARM1 inhibitor SGC2085 combined with ionizing radiation.7.Western Blot method:To detect the changes of apoptosis,cycle and adme-R,mme-R-related protein expression in colon cell.8.The effect of SGC2085 combined with ionizing radiation on autophagy in HCT116 and HT29 cells was detected by MDC method;DCFH-DA probe was used to detect the effect of SGC2085 combined with ionizing radiation on HCT116 and HT29 cell ROS.Rhodamine 123 and JC-1 were used to detect the changes of mitochondrial membrane potential of HCT116 and HT29 cells by SGC2085 combined with ionizing radiation.9.Changes in the expression of HCT116 and HT29 cell DNA damage-related proteins γ-H2AX and 53BP1 by immunofluorescence10.Statistical analysis of data using Graphpad prism 8.0 software,the groups of measuring data inx±s,normal and variance of data,using the single factor analysis of variance(One-Way ANOVA)to compare the indexes of differences between different groups,LSD method is used to compare between groups of two.P<0.05 was considered to be statistically significa.Results:1.Association of CARM1 with disease stage and survival in patients with COADThe results of bioinformatics analysis through the online analysis website showed that CARM1 was at a high expression level(P<0.01)in colon cancer tissues,and with the increase of the stage of colon cancer patients,the expression level of CARM1 gradually increased,and the survival of patients with high basal expression level of CARM1 was worse than that of lower basal expression in the middle and late stages(P=0.71).2.Effects of ionizing radiation on the expression of CARM1 and its regulated monomethyl arginine or asymmetric dimethyl arginine protein in colon cancer cells HCT116The protein expression level of CARM1 in colon cancer cells HCT116 after irradiation increased with the increase of irradiation dose.The changes of endogenous protein levels containing mme-R(monomethylarginine protein)in HCT116 and HT29 cells after irradiation were determined by Western Blot method,and it was found that the overall expression level of arginine monomethylation in the two colon cancer cells increased with the increase of irradiation intensity.3.Effect of SGC2085 on colon cancer cells CARM1.Compared with the control group,the protein expression level of colon cancer cells HCT116 after SGC2085 was significantly down-regulated.4.Effect of SGC2085 on endogenous levels of adme-R and mme-R on arginine residues in colon cancer cellsThe changes of endogenous protein levels containing mma-R(monomethylarginine protein)and adme-R(asymmetric dimethyl protein)in HCT116 after the action of SGC2085 were determined by Western Blot method,and it was found that the overall levels of arginine monomethylation and asymmetric arginine dimethylation in HCT116 cells decreased with the increase of SGC2085 concentration.5.Effect of CARM 1 inhibitor SGC2085 on radiation-induced colon cancer cell proliferation and survivalThe degree of proliferation inhibition of HCT116 and HT29 colon cancer cells increased with the increase of the concentration of SGC2085 and the increase of irradiation dose(P<0.01).Through the calculation of IC50,1.5 μM and 2.5 μM,4Gy,and 6Gy are the optimal inhibitory concentrations and doses for HCT116 and HT29 cells,respectively;the optimal action time was 24h;compared with the control group,the cell proliferation ability and colony formation ability of SGC2085 on colon cancer cells were inhibited(P<0.05),and the degree of inhibition was more obvious after irradiation(P<0.05).6.Effect of CARM 1 inhibitor SGC2085 on radiation-induced colon cancer cell migration and invasion capacityIn cell scratch experiment,HCT116 and HT29 cells were treated with SGC2085 for 48 h,and the cell migration capacity of SGC2085 group limited weakening compared with the control group(P<0.05).At 48 h after radiation,the migration capacity of cells in the SGC2085+IR group also decreased significantly(P<0.05).Transwell experiment:In the unirradiated HCT116 and HT29 cells,the relative invasion rate of SGC2085 was significantly reduced compared to the control group(P<0.05).At 48 h after irradiation,the invasion ability of cells Effectively inhibition of SGC2085 compared to the control group(P<0.05).7.Effect of the CARM1 inhibitor SGC2085 on radiation-induced colon cancer cell cycleCompared with the control group,there was obvious G0/G1 phase blockade(P<0.05)in the SGC2085 combined irradiation group in HCT116 and HT29 cells,and the detection of cycle-related proteins by Western Blot method found that the expression of P21 protein in the SGC2085 group had an upward trend in the unirradiated group of HCT116 and HT29 cells,and the expression trend of P21 protein increased more significantly after irradiation.8.Effects of CARM1 inhibitor SGC2085 on radiation-induced apoptosis in colon cancer cellsIn the two colon cancer cells,the SGC2085 group was able to increase the apoptosis rate of cells compared with the control group,and after radiation,the apoptosis rate of the combined group was significantly increased(P<0.01),which promoted the expression level of pro-apoptotic protein and protein related to the formation of apoptotic bodies.9.Effects of CARM1 inhibitor SGC2085 on radiation-induced autophagy in colon cancer cellsFlow cytometry was used to detect the effect of SGC2085 on the autophagy of cells induced by ionizing radiation on the autophagy of two colon cancer cells before and after radiation,and it was observed that colon cancer cells had a slight tendency to increase autophagy after 24 h and 48 h irradiation(P<0.01),the autophagy rate of SGC2085 increased significantly(P<0.05),and after radiation,the apoptosis rate of the combined group increased significantly compared with the negative control group(P<0.01);The MDC method was used to detect the changes of autophagy incolon cancer cells at 24 h after irradiation,which was consistent with the flow cytometry results,and incolon cancer cells,the autophagy rate of the SGC2085 group increased to a large extent compared with the control group,and the trend was more significant after irradiation(P<0.01).10.Effect of CARM1 inhibitor SGC2085 on mitochondrial function and ROS of radiation-induced colon cancer cellsIn both colon cancer cells,ROS production was increased in the irradiated group compared with cells in the unirradiated group,and membrane potential decreased.The ROS generation and membrane potential reduction of SGC2085 were significantly different from those in the negative control group at 3 h after combined irradiation(P<0.01).11.Effect of the CARM1 inhibitor SGC2085 on radiation-induced DNA damage in colon cancer cellsThe focal numbers of γ-H2AX and 53BP1 in colon cancer cells at different time points after irradiation were detected by immunofluorescence,and it was found that the focal numbers of y-H2AX and 53BP1 increased significantly compared with the unirradiated group at 3 h and 6 h after irradiation,and the y-H2AX and 53BP1 focal formation in the combined group were more significant,that is,SGC2085 enhanced X-ray significantly increased the apoptosis level of DNA double-strand break damage in colon cancer cells.This change is statistically significant(P<0.01).Conclusion:1.Ionizing radiation can induce the expression of CARM1 protein in colon cancer cells;2.Ionizing radiation can promote CARM1-induced panthenine monomethylation or ubiquinone arginine asymmetric dimethylation;3.The CARM1 inhibitor SGC2085 can reduce the proliferative activity of HCT116 and HT29 cells,increase the level of autophagy and DNA damage,cause G0/G1 phase arrest and induce apoptosis.4.SGC2085 can promote the increase of mitochondria-dependent ROS in HCT116 and HT29 cells,thereby inducing elevated autophagy level;5.SGC2085 can effectively improve the radiation sensitivity of colon cancer cells. |
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