UBQLN2通过上调嘌呤代谢水平诱导食管鳞癌细胞放射抵抗的实验研究

Experimental study on UBQLN2 induced-radioresistance in esophageal squamous cell carcinoma cells by upregulating purine metabolism levels

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。

ObjectiveTo 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.MethodssiRNA 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.ResultsCompared 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.ConclusionThe expression level of UBQLN2 was negatively related to the radiosensitivity of ESCC by up-regulating purine metabolism.