Study On The Mechanism Of Radioresistance Regulated By Glycosyltranferase GALNT2 In Lung Adenocarcinoma

Background: Lung cancer is the malignant tumor with the highest morbidity and mortality in China and the world.Lung adenocarcinoma is the most common histologic subtype of lung cancer,with an average 5-year survival rate of approximately 15%.The onset of lung adenocarcinoma is insidious,and most patients are locally advanced or have distant metastasis when seeking treatment,often losing the opportunity of radical surgery.Therefore,radiotherapy is the main method to control the inoperable early and unresectable locally advanced lung adenocarcinoma clinically.However,the biggest obstacle to radiotherapy is the radioresistance of tumor cells,which is also an important factor leading to the failure of radiotherapy for lung adenocarcinoma and the death of patients.Glycosyltransferase GALNT2 is the initiator of O-glycan synthesis,abnormal expression of GALNT2 plays a key role in the occurrence and development of a variety of tumors,affecting tumor cell proliferation,adhesion,invasion,metastasis and other malignant phenotypes.However,the relationship between GALNT2 and radioresistance of lung adenocarcinoma remains unclear.Objective: The purpose of this study was to study the role of GALNT2 in radioresistance of lung adenocarcinoma,and to explore the molecular mechanism of GALNT2 in the regulation of radioresistance of lung adenocarcinoma.Methods:(1)Oncomine and TCGA databases were used to analyze the expression of GALNT2 m RNA in lung adenocarcinoma tissues,Immunohistochemical technique was used to detect the expression of GALNT2 protein in lung adenocarcinoma specimens,and survival analysis was performed.(2)The differential expression of GALNT2 was analyzed by i TRAQ quantitative protein mass spectrometry,and verified by Western blot.(3)GALNT2 overexpressing lentivirus was transfected into parent cells of lung adenocarcinoma,and GALNT2 RNA interfering lentivirus was transfected into radioresistant cells.The effects of GALNT2 expression changes on radioresistance of lung adenocarcinoma cells were analyzed by clonal formation test and flow cytometry.(4)The targeted modified glycoproteins of GALNT2 were screened by mass spectrometry and lectin pull-down techniques,and verified by functional rescue experiments.(5)The down regulated GALNT2 expression cells were injected subcutaneously into nude mice to establish the nude mice model of lung adenocarcinoma,tumor size and growth were monitored,and tumor growth curve was drawn,the difference of radiosensitivity was compared after radiation treatment.Results:(1)Oncomine and TCGA analysis showed that GALNT2 m RNA was significantly overexpressed in lung adenocarcinoma tissues compared with normal tissues.Immunohistochemical results showed that the expression level of GALNT2 protein in lung adenocarcinoma tissues was significantly higher than that in adjacent tissues.Survival analysis showed that patients with high GALNT2 expression had a shortened overall survival and poor prognosis.(2)Quantitative proteomics was used to screen and verify the results by Western blot.Meanwhile,CAZY,GGDB and KEGG Glycan databases were searched and analyzed.It was found that GALNT2 was the only glycotransferase with increased expression in radioresistant lung adenocarcinoma cells.(3)In radioresistant lung adenocarcinoma cells,when GALNT2 expression was down-regulated,the clonal-form ability of the cells was decreased,the apoptotic ratio and G2/M phase cells were increased,and radioresistance was decreased.In lung adenocarcinoma parent cells,when GALNT2 expression was upregulated,the clonal-form ability of the cells was increased,the apoptotic ratio and G2/M phase cells were decreased,and radioresistance was increased.(4)The lectin VVA,which specifically recognized O-glycan,combined with mass spectrometry,was used to pull down glycoproteins.it was found that IGF-1R had O-glycosylation modification.Overexpression of GALNT2 can increase the O-glycosylation level of IGF-1R.Inhibition of GALNT2 can decrease the level of Oglycosylation of IGF-1R.Meanwhile,silencing IGF-1R reversed GALNT2-mediated radioresistance in lung adenocarcinoma cells.(5)Inhibition of GALNT2 expression can slow down the growth,reduce the volume,weight and increase the radiosensitivity of nude mice.Conclusion:(1)GALNT2 was highly expressed in lung adenocarcinoma and was closely related to the poor prognosis of patients.(2)Both in vivo and in vitro experiments confirmed that GALNT2 could regulate the radioresistance of lung adenocarcinoma.(3)GALNT2 mediated radioresistance of lung adenocarcinoma by promoting O-glycosylation of IGF-1R.In conclusion,this study reveals a new mechanism by which GALNT2 regulates radiosensitivity of lung adenocarcinoma from clinical data analysis,cell studies and in vivo animal experiments.Our findings can provide a new target for overcoming radiosensitivity of lung adenocarcinoma and a scientific basis for radiosensitivity prediction and individualized radiotherapy of lung adenocarcinoma.It has important theoretical significance and clinical translational value.