Collagen Type Ⅰ Alpha 1 Chain Influences The Radiosensitivity Of Glioblastoma Cells In Hypoxia

Objective:Glioblastoma（GBM）was a highly hypoxia tumor,and hypoxia was an important factor in the development of tumor radiation resistance.The purpose of this study was to investigate the effect of COL1A1 on radiosensitivity of hypoxia GBM cells and to explore its potential mechanism.Methods:The expression of COL1A1 gene in GBM was analyzed by the Oncomine database.The RNA-seq data of the GBM cohort and the clinical data of patients were obtained from the public database.Kaplan-Meier method was used to evaluate the prognosis.The hypoxia gene set was obtained from the molecular signature database（Molecular Signatures Database）,and the correlation between COL1A1 and hypoxia gene was identified by co-expression analysis.After irradiation and hypoxia（1%O₂）treatment,the expression of COL1A1 in GBM cells was detected by real-time fluorescence quantitative PCR and Western blotting.The lentivirus vector containing COL1A1 overexpression and COL1A1 sh RNA was constructed,and the glioma cell line with stable overexpression and knockdown of COL1A1 was established by lentivirus transfection.CCK-8 kit was used to analyze the proliferation of hypoxia and normoxia GBM cells.Flow cytometry was used to evaluate the apoptosis of hypoxia and normoxia GBM cells.Plate colony formation test was used to evaluate the radiosensitivity of GBM cells.Autophagy was evaluated by m RFP-GFP-LC3 adenovirus infection by fluorescence microscope detection.Western blotting was used to detect the expression of apoptosis and autophagy-related proteins（PARP,LC3B,P62）,and DNA repair proteinγ-H2Ax.Results:1.COL1A1 gene is generally highly expressed in GBM,and the results of survival analysis suggest that it is significantly related to the short survival of GBM（TCGA:P=0.045;CGGA:P=0.008）.2.In TCGA and CGGA database,co-expression analysis showed that 16common hypoxia genes were significantly correlated with COL1A1 expression.It is speculated that COL1A1 can be used as a hypoxia-related gene,and its expression level may be hypoxia-dependent.3.The expression of COL1A1 m RNA in GBM cells increased after 8Gy X-ray irradiation.In a certain period of time,continuous hypoxia can cause the expression of COL1A1 to increase progressively.4.Stable overexpression of COL1A1 can effectively up-regulate the expression of COL1A1 in glioblastoma cells,while stable transfection of COL1A1sh RNA can effectively down-regulate the expression of COL1A1 in glioblastoma cells.5.The results of CCK-8 assay showed that overexpression of COL1A1 could promote cell growth under both normoxia and hypoxia conditions,but the effect of promoting cell proliferation was more significant under hypoxia conditions（normoxia condition:p<0.01,hypoxia condition:P<0.0001）.Knockdown of COL1A1 significantly inhibited the growth activity of hypoxia M059K cells（p<0.0001）,but there was no significant difference in cell viability between the two groups under normoxia conditions.6.Under normoxia condition,there was no significant difference in the proportion of apoptosis both the overexpression of COL1A1 group and the knockout COL1A1 group compared with the control group.Under hypoxia condition,the apoptosis ratio of U251MG cells was significantly decreased after overexpression of COL1A1（P=0.0312）.After knockout of COL1A1,the apoptosis rate of M059K cells was not significantly different from that of the control group,but the apoptosis rate still showed an increasing trend.After 8Gy X-ray irradiation,overexpression of COL1A1 could significantly inhibit the apoptosis of GBM cells（normoxia condition:P=0.0008,hypoxia:P=0.0009）,while the apoptosis rate after knockout of COL1A1 was significantly higher than that in the negative control group（normoxia condition:P=0.0001,hypoxia condition:p<0.0001）.7.Plate clone formation assay showed that COL1A1 could regulate the radiosensitivity of hypoxia GBM cells.Knockdown COL1A1 inhibited the radiosensitivity of hypoxia M059K cells（SER=1.39）.Overexpression of COL1A1increased the radioresistance（SER=0.77）of hypoxia U251MG cells,but the radioresistance induced by COL1A1 was not obvious under normoxia conditions.8.Under hypoxia conditions,knockdown of COL1A1 decreased the autophagy level of M059K cells,while overexpression of COL1A1 could promote autophagy of U251MG cells.Conclusion:Abnormally high expression of COL1A1 in glioblastoma patients is significantly associated with poor prognosis.Bioinformatics analysis suggests that COL1A1 may be a hypoxia-related gene.Radiation injury and hypoxia can increase the expression of COL1A1 in human glioblastoma cells.Overexpression of COL1A1 can promote the proliferation and radiation resistance of hypoxia glioblastoma cells,while knockdown COL1A1 can significantly inhibit cell proliferation and radiation resistance.It may be related to the regulation of apoptosis and autophagy of hypoxia tumor cells and can be used as a molecular target for radiosensitization in further research.