CPT1A Mediates Radiation Sensitivity In Colorectal Cancer Through Down Regulating FOXM1-SOD1/SOD2/CAT Axis

Purpose and Background:Colorectal cancer(CRC)is a malignant tumor with high morbidity and high mortality worldwide.The incidence of colorectal cancer in China has increased year by year and has leaped to the third place.Patients with locally advanced(cT3-4N+)rectal cancer adopt the standard treatment strategy of neoadjuvant chemoradiotherapy.Radiotherapy can be used in the condition of initial unresectable,postoperative residual and recurrence,oligo-metastasis and others types of colon cancer,which enables patient benefit from it.However,there is no effective method to solve the problem of tumor recurrence and metastasis caused by radiation resistance.Therefore,it is urgent to reveal the molecular mechanism of radiation resistance of colorectal cancer to strengthen the effect of radiotherapy and improve the prognosis of CRC patients.Experimental Design:A high-throughput and rapid screening of radiation-related genes was performed using the lipid-related gene shRNA virus library.The qPCR,western blot,and immunohistochemistry were used to clarify the expression of CPT1A in colorectal cancer tissues and immunohistochemistry combined with TRG score were used to explore the correlation between CPT1A and neoadjuvant treatment efficiency.The correlation between CPT1A and radiation sensitivity was clarified by stable knockout/overexpression of CPT1A,colony-forming assay,multi-target single-hit model generation,comet assay,detection of y-H2A.X expression,and in vivo experiments.Transcriptome sequencing,bioinformatic analysis,reactive oxygen species(ROS)detection,glutathione(GSH)/oxidized glutathione(GSSG)ratio detection,enzyme activity detection,qPCR,western blot,and other experiments were used to reveal the molecular mechanism of radio-sensitivity induced by CPT1 A.Results:By using the lipid-related gene shRNA virus library,a mouse radiation-sensitive gene Cptla was identified.The results of protein sequence alignment showed that mouse Cpt1a was quite similar to human CPT1A.Bioinformatic analysis,RT-qPCR,western blot and immunohistochemical staining revealed that CPT1A was downregulated in CRC.Immunohistochemical staining and correlation analysis of rectal cancer tissues suggested that CPT1A is negatively correlated with the efficiency of neoadjuvant chemoradiotherapy.Afterwards,the correlation between human CPT1A gene and radiation sensitivity was explored using human colorectal cancer cell lines with stable knockout/overexpression of CPT1 A.CPT1A was shown to be a radiation-sensitive gene by colony-forming assay,multi-target single-hit model generation,comet assay,and detection of γ-H2A.X expression.The aforementioned results can be replicated and rescued in radiation-resistant cell line HCT-15-25F.Transcriptome sequencing combined with bioinformatic analysis indicated that the peroxisome pathway is an important downstream pathway of CPT1 A.The ROS content,GSH/GSSG ratio,SOD enzyme activity,and CAT enzyme activity were detected in CPT1A stable knockout/overexpression cell lines and radiation-resistant cell line HCT-15-25F,intracellular and under 6 Gy radiation.CPT1A was found to regulate the intracellular GSH/GSSG ratio,SOD enzyme activity and CAT enzyme activity.Using RT-qPCR and western blot,it’s found that CPT1A downregulates the mRNA and protein levels of SOD1,SOD2,and CAT.Bioinformatic analysis,RT-qPCR,and western blot suggested that the transcription factor FOXM1 is a bridge among CPT1A and other three enzymes.In a word,CPT1A enhanced ROS scavenging-mediated radio-sensitivity in colorectal cancer via FOXM1-SOD1/SOD2/CAT axis.Conclusion:Our results indicate that CPT1A is often downregulated in CRC,and it leads to the increased mRNA and protein levels of FOXM1,promotes the transcription and translation of SOD1,SOD2,and CAT,promotes ROS scavenging,and eventually leads to cell radiation resistance.Our study unravels the functional mechanism of CPT1A mediating radiation sensitivity in colorectal cancer through increased ROS scavenging via FOXM1-SOD1/SOD2/CAT axis.This study leads to a novel treatment strategy targeting radiotherapy resistance.