Study On The Effect Of Glutamine Synthetase On The Sensitivity Of Radiotherapy For Hepatocellular Carcinoma

BACKGROUND:Hepatocellular carcinoma(HCC)is the most common primary liver cancer,and radiation therapy is increasingly used in the treatment of liver cancer because of its safe and non-invasive nature.However,due to the inherent radioresistance of hepatocellular carcinoma and the low radiation tolerance of the surrounding normal liver,the treatment efficiency of radiation therapy for HCC is low.Therefore,strategies related to increasing tumor radiosensitivity and reducing normal tissue complications are urgently needed to improve the efficacy of radiotherapy for HCC.Autophagy and reactive oxygen species(ROS)are important factors affecting tumor radiosensitivity,and the tumor therapeutic target glutamine synthetase(GS)is highly expressed in HCC which can affect the levels of autophagy and ROS through corresponding pathways.Therefore,targeting GS may improve the sensitivity of HCC radiation therapy by regulating autophagy and ROS levels.METHODS:This study used bioinformatics to perform differential GS expression analysis in several databases,including TCGA(The Cancer Genome Atlas),ICGC(International Cancer Genome Consortium)and GEO(Gene Expression Omnibus).The proliferation efficiency of cells was examined by CCK-8,colony formation assay to determine the effect of glutamine(GLN),GS inhibitor(LMethionine Sulfoximine,MSO)and Ionizing Radiation(IR)on HCC proliferation.The effects of MSO on GS expression in HCC cells were evaluated by real-time fluorescence quantitative PCR(qPCR)and protein immunoblotting assay(Western Blot,WB).The inhibition efficiency of MSO on GS viability was examined by GS assay kit.The effects of MSO and GLN on HCC cell migration were evaluated by scratch assay.The effects of MSO and IR on HCC cell migration were evaluated by ROS assay kit.MSO and IR on the level of ROS in HCC cells and tissues.The effect of MSO and IR on the level of autophagy in HCC cells was determined using MDC staining and WB experiments.Finally,an in vivo ectopic xenograft tumor model was established using HepG2 cells to determine the inhibitory effects of MSO and IR on tumor growth.RESULTS AND CONCLUSIONS:In this study,analysis of TCGA,ICGC and GEO databases revealed that GS was highly expressed in HCC tissues and could effectively predict the distinction between tumor and normal liver tissues.Although the GS inhibitor MSO induced a negative feedback regulation in HCC cells resulting in increased GS-mRNA and protein expression levels,it did not affect cell proliferation.However,in the absence of GLN,MSO treatment further reduced the diminished cell proliferation caused by GLN deficiency,suggesting that endogenous GLN levels are a direct regulator of HCC proliferation.In addition,the migration ability of HCC cells was diminished in the absence of GLN,and MSO further enhanced migration inhibition,suggesting that GS is a potential therapeutic target for HCC.In this paper,we also investigated the effect of MSO on HCC radiotherapy.In vitro cellular assays demonstrated that radiation-treated HCC cells showed some radioresistance.However,combined MSO increased the sensitivity of HCC cells to radiation treatment.Further studies showed that MSO treatment increased ROS in HCC cells and inhibited autophagic flow processes,suggesting that MSO could increase the radiation killing effect on HCC cells through these pathways.In vivo experiments also showed that the combination of MSO and IR could inhibit tumor growth more effectively.In conclusion,this thesis focuses on GS-mediated radioresistance of HCC and reveals the role of MSO in influencing HCC radiosensitivity to radiation therapy by regulating ROS,autophagy levels.