| 马艳丽,马蓉,王嘉琳,等.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[J].中华放射医学与防护杂志,2024,44(11):909-916.Ma Yanli,Wang Jialin,Ma Rong,et al.Experimental study on UBQLN2 induced-radioresistance in esophageal squamous cell carcinoma cells by upregulating purine metabolism levels[J].Chin J Radiol Med Prot,2024,44(11):909-916 |
| 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 |
| 投稿时间：2024-02-20 |
| DOI：10.3760/cma.j.cn112271-20240220-00065 |
| 中文关键词: 人食管鳞癌细胞 UBQLN2 放射敏感性 嘌呤代谢 |
| 英文关键词:Esophageal squamous cell carcinoma (ESCC) UBQLN2 Radiosensitivity Purine metabolism |
| 基金项目:国家自然科学基金(82060433);宁夏自然科学基金(2021AAC05018) |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| Objective 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. |
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