The Function And Molecular Mechanism Study Of Circ-CPSF1 In Caenorhabditis Elegans Radiation Injury Model

Background and ObjectiveRadiation therapy is one of the major methods for cancer patients.It’s undeniable that radiation therapy also cause damage to normal cells,known as radiation injury or radiotherapy injury.Ionizing radiation injury leads to poor clinical treatment effects and poor patient prognosis.For example,during radiation therapy for left-sided breast cancer patients,the heart is at risk of exposure to ionizing radiation,causing radiation-induced heart disease(RIHD),which has a serious impact on the patient’s quality of life.Recently,circRNA has been researched extensively.Based on its unique circular structure,it has broad prospects in cell function and biomedical applications.Compared with other model organisms such as mice,C.elegans is an excellent model organism for radiation damage research because of its convenient culture,good stability,fast passage,low cost,and sensitivity to radiation.Since circular RNAs are well conserved among different species,and taking advantage of the genetic advantages of C.elegans,our study aims to screen the target gene circ-CPSF1 by constructing a radiation-induced C.elegans model,and initially study the biological effect and molecular mechanism of circ-CPSF1 in ionizing radiation,as to clarify the potential relationship between circ-CPSF1 and ioniing radiation damage.It should shed ligh on the future application of circRNA in ionizing radiation damage research.Materials and Methods1、RNA sequencing technology was applied between the C.elegans before and after ionizing radiation.Then we screened out the F19B10.4 gene in combination with radiation-related databases,and F19B10.4 gene was knocked out by CRISPRCas9 technology to obtain knockout strains M263,M264,and M265.Biological verifications were performed to make sure ionizing radiation damage C.elegans model holds.2、circRNA sequencing technology was performed between knockout nematodes and wild-type nematodes.Then we screened out the target gene circCPSF1 and circ-CPSF1 recombinant plasmid was constructed to interfere with circCPSF1 expression specifically.Biological studies such as lifespan,the number of eggs,germ cell apoptosis,and time of embryonic development were conducted for further verification,Log-rank(Mantel-Cox)was uesd to analyze the survival curve of nematodes,one-way analysis of variance(ANOVA)was used to study the reproductive status of nematodes.3、ROS fluorescent probe and RT-qPCR technology were used to detect the regulatory effect of circ-CPSF1 on oxidative stress pathway in C.elegans exposed to ionizing radiation;JC-1 mitochondrial membrane potential detection was used to explore the regulatory mechanism of circ-CPSF1 on mitochondrial damage in C.elegans exposed to ionizing radiation.The mechanism of circ-CPSF1 in the apoptosis pathway of C.elegans exposed to ionizing radiation was analyzed by detecting the expression of apoptosis-related genes,Graphing and data analysis were performed using IBM SPSS Statistics 20 and GraphPad Prism 8.0 software.Results1、We screened out the radiation-related gene F19B10.4 in C.elegans with and without X-ray exposure by sequencing technology and bioinformatics analysis.F19B10.4 gene was up-regulated exposed to ionizing radiation in C.elegans.Compared with the wild-type C.elegans and knockout strain,there was no significant difference in growing.However,the growth of the knockout strain was significantly inhibited by ionizing radiation.The above results indicated that the F19B 10.4 knockout strain can be used as an ionizing radiation damaged nematode model.2、Compared with the control group and the normal group,the circ-CPSF1 RNAi.group had longer lifespan,more eggs,less apoptotic germ cells and shorter development time of embryogenesis exposed to ionizing radiation.3、Through the detection of ROS fluorescent probe and the analysis of the expression of oxidative stress-related genes,it was found that the fluorescence intensity of the circ-CPSF1 RNAi group was weakened compared with the control group and the normal group after ionizing radiation,the mitochondrial membrane potential of the circ-CPSF1 RNAi group increased more seriously.The results of the expression of DNA damage-related genes husl,clk-2,mrt-2 in the circ-CPSF1 RNAi group was lower than that in the control and normal groups exposed to ionizing radiation.The results of the expression of apoptosis-related genes showed that the expression of pro-apoptotic genes cep-1,egl-1,ced-13,ced-4,ced-3 in the circCPSF1 RNAi group was lower than that in the control and normal groups exposed to ionizing radiation,and the anti-apoptotic gene ced-9 in the circ-CPSF1 RNAi group were higher than that of control group and normal group.Conclusion1、Circ-CPSF1 can aggravate the damage of ionizing radiation on lifespan,the number of eggs,germ cell apoptosis,and time of embryonic development of C.elegans exposed to ionizing radiation.2、circ-CPSFl can aggravate ionizing radiation damage to C.elegans through oxidative stress pathway,DNA damage repair pathway and apoptosis pathway.