摘要目的:观察泛素样蛋白2(UBQLN2)表达水平对人食管鳞癌(ESCC)细胞EC109和KYSE30放射敏感性的影响，并探索其分子机制。方法:采用小RNA干扰和慢病毒转染技术构建UBQLN2敲低和过表达细胞系，给予4 Gy X射线照射。CCK-8、克隆形成实验检测UBQLN2表达水平对ESCC放射敏感性的影响。采用UPLC-Q－TOF-MS技术进行代谢物差异分析及京都基因与基因组百科全书数据库(KEGG)通路分析。qRT-PCR实验验证代谢组学结果。通过加入代谢酶抑制剂和外源性补充代谢物进一步验证UBQLN2表达水平对ESCC放射敏感性的影响。结果:与单纯照射相比，下调ESCC细胞系中UBQLN2的表达水平再给予照射，可使EC109和KYSE30细胞存活率分别下降32.29%和16.42%( t＝5.35、4.88， P<0.05)，克隆形成率分别下降11.07%和7.47%( t＝4.18、5.09， P<0.05)。而上调UBQLN2的表达水平可使EC109和KYSE30细胞存活率上升14.07%和10.64%( t＝5.88、4.21， P<0.05)，克隆形成率上升6.53%和7.87%( t＝8.60、8.26， P<0.05)。代谢组学分析显示，UBQLN2表达水平改变主要影响嘌呤代谢通路。qRT-PCR实验显示，UBQLN2表达水平与5种嘌呤代谢酶的mRNA水平呈正相关。在UBQLN2过表达ESCC细胞系中加入霉酚酸(MPA)后，细胞存活率分别下降了18.28%和25.58% ( t＝7.76、10.95， P<0.05)，克隆形成率分别下降了9.33%和9.93%( t＝5.97、8.02， P<0.05)。而在UBQLN2敲低细胞系中外源性补充核苷酸后细胞的存活率上升了8.28%和10.74%( t＝2.83、6.20， P<0.05)，克隆形成率分别上升了7.33%和5.80%( t＝7.16、5.49， P<0.05)。 结论:UBQLN2表达水平与ESCC的放射敏感性呈负相关。UBQLN2可能通过上调嘌呤代谢水平参与ESCC放射敏感性的调控。

abstractsObjective:To observe the effect of ubiquilin 2 gene (UBQLN2) on the radiosensitivity of human esophageal squamous cell carcinoma cells (ESCC) of EC109 and KYSE30, and explore underlying molecular mechanism.Methods:siRNA and lentivirus transfection techniques were used to establish UBQLN2-knockdown and UBQLN2-overexpression cell lines. The cells were irradiated with a dose of 4 Gy X-rays. UPLC-Q-TOF-MS technique was used for metabolite difference analysis and KEGG pathway analysis. The results of metabonomics analyses were verified by qRT-PCR assay. The influence of UBQLN2 level on the radiosensitivity of ESCC was confirmed by CCK-8 cell proliferation assay and clone formation assay and further verified by treating the cells with metabolic enzyme inhibitors and exogenous metabolites.Results:Compared with irradiation alone, down-regulating UBQLN2 in EC109 and KYSE30 cell lines reduced the cell survival by 32.29% and 16.42% ( t=5.35, 4.88, P<0.05), and reduced the clone formation rate by 11.07% and 7.47% after 4 Gy irradiation, respectively ( t=4.18, 5.09, P<0.05). On the contrary, up-regulating UBQLN2 in EC109 and KYSE30 cell lines increased the survival rate by 14.07% and 10.64% ( t=5.88, 4.21, P<0.05), and increased the clone formation rate by 6.53% and 7.87% after 4 Gy irradiation, respectively ( t=8.60, 8.26, P<0.05). Metabonomics study showed that the purine metabolic pathway was significantly enriched after down-regulating UBQLN2 in EC109 cell. The qRT-PCR experiment showed a positive correlation between the expression level of UBQLN2 and the mRNA level of five purine metabolism enzymes. The viability of irradiated UBQLN2-overexpression cells decreased by 18.28% and 25.58%, respectively ( t=7.76, 10.95, P<0.05), and the clone formation rate decreased by 9.33% and 9.93%, respectively ( t=5.97, 8.02, P<0.05) after adding mycophenolic acid(MPA). However, the survival rate of cells increased by by 8.28% and 10.74% ( t=2.83, 6.20, P<0.05), and the clone formation rate increased by 7.33% and 5.80%, respectively ( t=7.16, 5.49, P<0.05), when exogenous supplementation of nucleotides (ATP + GTP) were added. Conclusion:The expression level of UBQLN2 was negatively related to the radiosensitivity of ESCC by up-regulating purine metabolism.