中国临床药理学杂志

代谢酶PGK1琥珀酰化修饰位点突变载体构建及其在HEK-293T细胞中表达的研究

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王瑶瑶 ^1a , 刘立琨 ^1b , 高秀丽 ^1b , 朱文斌 ^1b , 郝经伟 ^1a , 岳丽玲 ^1b

中国临床药理学杂志 | 临床与基础桥接研究 2025,41(9): 1229-1235

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中国临床药理学杂志 |临床与基础桥接研究 2025 , 41 (9) : 1229 -1235

代谢酶PGK1琥珀酰化修饰位点突变载体构建及其在HEK-293T细胞中表达的研究

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王瑶瑶^1a, 刘立琨^1b, 高秀丽^1b, 朱文斌^1b, 郝经伟^1a, 岳丽玲^1b

作者信息

^1a.齐齐哈尔医学院，医学技术学院，黑龙江齐齐哈尔 161006

^1b.齐齐哈尔医学院，医药科学研究院，黑龙江齐齐哈尔 161006

通讯作者:

岳丽玲，教授，博士生导师 Tel: （0452）2663715 E-mail: yuell1025@126.com

作者简介:

王瑶瑶（1994-），女，硕士研究生，主要从事肿瘤发病机制的相关研究

Research on the construction of metabolizing enzyme PGK1 succinylation modification site mutation vector and its expression in HEK-293T cells

Yao-yao WANG^1a, Li-kun LIU^1b, Xiu-li GAO^1b, Wen-bin ZHU^1b, Jing-wei HAO^1a, Li-ling YUE^1b

Affiliations

^1a.School of Medical Science and Technology, Qiqihar Medical University, Qiqihar 161006, Heilongjiang Province, China

^1b.Research Institute of Medicine and Pharmacy, Qiqihar Medical University, Qiqihar 161006, Heilongjiang Province, China

出版时间: 2025-05-17 doi: 10.13699/j.cnki.1001-6821.2025.09.006

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摘要

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目的

通过构建磷酸甘油酸激酶1（PGK1）过表达载体（PGK1-WT）及4个赖氨酸位点琥珀酰化突变载体，实现其在人胚肾HEK-293T细胞中过表达并筛选PGK1琥珀酰化关键位点。

方法

用GEPIA2数据库分析正常乳腺组织（291例）和乳腺癌组织（135例）中PGK1 mRNA的表达情况，以及PGK1表达水平与乳腺癌患者总生存率的关系；用蛋白质修饰组学检测4株三阴性乳腺癌（TNBC）细胞系（MDA-MB-436、MDA-MB-468、MDA-MB-231、BT-549）及正常乳腺上皮细胞（MCF-10A）中蛋白质琥珀酰化水平；用点突变试剂盒构建K11、K131、K323、K361琥珀酰化正、负向突变载体。实验分为NC组、shPGK1-496组、shPGK1-220组、PGK1-WT组及K11、K131、K323、K361位点正、负向突变体组。用实时荧光定量聚合酶链反应检测NC组、shPGK1-496组和shPGK1-220组的PGK1 mRNA表达情况；用蛋白质印迹法检测NC组、shPGK1-496组和shPGK1-220组PGK1蛋白的表达情况及NC组、PGK1-WT组及4个位点正、负向突变体组的Flag表达情况，并通过免疫共沉淀检测PGK1琥珀酰化关键位点。

结果

与正常乳腺组织相比，PGK1 mRNA水平在乳腺癌组织中高表达，且PGK1高表达的乳腺癌患者生存期较短。TNBC细胞中PGK1的4个赖氨酸位点呈高琥珀酰化。NC组、shPGK1-496组和shPGK1-220组的PGK1 mRNA相对表达水平分别为1.03±0.23、0.15±0.11和1.42±0.69，PGK1蛋白相对表达水平分别为1.08±0.05、0.49±0.05和1.28±0.01，shPGK1-496组的上述指标与NC组比较，在统计学上差异均有统计学意义（均P<0.01）。NC组、PGK1-WT组及K11、K131、K323位点正、负向突变体组的Flag蛋白相对表达水平分别为0、3.43±0.09、3.28±0.10、2.28±0.11、3.03±0.03、2.79±0.11、3.29±0.09和2.98±0.04，PGK1-WT组及K11、K131、K323位点正、负向突变体组与NC组比较，在统计学上差异均有统计学意义（均P<0.01）。NC组、PGK1-WT组及K361正、负向突变体组的Flag蛋白相对表达水平分别为0、2.58±0.11、3.04±0.02和4.04±0.10，PGK1-WT组及K361正、负向突变体组与NC组比较，在统计学上差异均有统计学意义（均P<0.01）。IgG组、PGK1-WT组及K11、K131、K323、K361位点负向突变体组的赖氨酸琥珀酰化水平分别为0、1.53±0.12、1.30±0.18、1.29±0.09、1.23±0.03和0.89±0.16，K361负向突变体组与PGK1-WT组比较，在统计学上差异有统计学意义（P<0.01）。

结论

TNBC细胞中PGK1 4个赖氨酸位点呈高琥珀酰化，且K361位点是其琥珀酰化的关键位点。

关键词

磷酸甘油酸激酶1 / 琥珀酰化 / 突变体 / 翻译后修饰

Abstract

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Objective

To achieve overexpression of phosphoglycerate kinase 1 (PGK1) in HEK-293T cells and screen the key site of its succinylation through constructing PGK1 overexpression vector (PGK1-WT) and four lysine site succinylation mutation vectors.

Methods

GEPIA2 database was used to analyze the expression of PGK1 mRNA in normal breast tissue (291 cases) and breast cancer tissue (135 cases) and the relationship between PGK1 expression level and the overall survival rate of breast cancer patients. Protein modification proteomics was used to detect the levels of protein succinylation in four triple negative breast cancer (TNBC) cell lines (MDA-MB-436, MDA-MB-468, MDA-MB-231, BT-549) and normal breast epithelial cells (MCF-10A); the succinylated positive and negative mutation vectors of K11, K131, K323, K361 were contructed with point mutation kit. The experiment was divided into NC, shPGK1-496, shPGK1-220, PGK1-WT groups and positive and negative mutant groups of K11, K131, K323 and K361. Real-time fluorescence quantitative polymerase chain reaction was used to detect the level of expression of PGK1 mRNA in NC, shPGK1-496 and shPGK1-220 groups. The expression levels of PGK1 proteins in NC, shPGK1-496 and shPGK1-220 groups, and the expression levels of Flag proteins in NC, PGK1-WT and four sites in positive and negative mutant groups were detected by Western blot. The key site of PGK1 succinylation was detected by co-immunoprecipitation.

Results

Compared with normal breast tissues, the level of PGK1 mRNA was highly expressed in breast cancer tissue, and the survival time of breast cancer patients with high PGK1 expression was shorter. Four lysine sites of PGK1 in TNBC cells were hypersuccinylated. The relative expression levels of PGK1 mRNA in NC, shPGK1-496 and shPGK1-220 groups were 1.03±0.23, 0.15±0.11 and 1.42±0.69, respectively; the relative expression levels of PGK1 protein were 1.08±0.05, 0.49±0.05 and 1.28±0.01 respectively; compared with NC group, the above indexes in shPGK1-496 group were statistically different (all P<0.01). The relative expression levels of Flag protein in NC group, PGK1-WT group and positive and negative mutant groups of K11, K131, K323 site were 0, 3.43±0.09, 3.28±0.10, 2.28±0.11, 3.03±0.03, 2.79±0.11, 3.29±0.09 and 2.98±0.04, respectively; compared with the NC group, PGK1-WT group and the positive and negative mutant groups of K11, K131 and K323 had statistical differences (all P<0.01). The relative expression levels of Flag protein in NC group, PGK1-WT group and K361 positive and negative mutant group were 0, 2.58±0.11, 3.04±0.02 and 4.04±0.10, respectively; compared with NC group, PGK1-WT group and K361 positive and negative mutant group had statistical significance (all P<0.01). The levels of lysine succinylation in IgG group, PGK1-WT group and negative mutant groups of K11, K131, K323, K361 were 0, 1.53±0.12, 1.30±0.18, 1.29±0.09, 1.23±0.03 and 0.89±0.16, respectively; compared with PGK1-WT group, the negativ mutant group of K361 had statistical significance (P<0.01).

Conclusion

The four lysine sites of PGK1 in TNBC cells are highly succinylated, and K361 site is critical to their succinylation.

Key words

phosphoglycerate kinase 1 / succinylation / mutant / post-translational modification

引用本文

王瑶瑶, 刘立琨, 高秀丽, 朱文斌, 郝经伟, 岳丽玲. 代谢酶PGK1琥珀酰化修饰位点突变载体构建及其在HEK-293T细胞中表达的研究. 中国临床药理学杂志, 2025 , 41 (9) : 1229 -1235 . DOI: 10.13699/j.cnki.1001-6821.2025.09.006

Yao-yao WANG, Li-kun LIU, Xiu-li GAO, Wen-bin ZHU, Jing-wei HAO, Li-ling YUE. Research on the construction of metabolizing enzyme PGK1 succinylation modification site mutation vector and its expression in HEK-293T cells[J]. Chinese Journal of Clinical Pharmacology, 2025 , 41 (9) : 1229 -1235 . DOI: 10.13699/j.cnki.1001-6821.2025.09.006

基金

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国家自然科学基金资助项目(81972491)
黑龙江省自然科学基金资助项目(H2022H108)
黑龙江省博士后面上基金资助项目(LBH-Z22295)
齐齐哈尔医学院研究生创新基金资助项目(QYYCX2022-17)

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2025年第41卷第9期

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文章信息

doi: 10.13699/j.cnki.1001-6821.2025.09.006

接收时间：2024-12-23
首发时间：2026-08-04
出版时间：2025-05-17

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收稿日期：2024-12-23

基金

国家自然科学基金资助项目(81972491)

黑龙江省自然科学基金资助项目(H2022H108)

黑龙江省博士后面上基金资助项目(LBH-Z22295)

齐齐哈尔医学院研究生创新基金资助项目(QYYCX2022-17)

作者信息

^1a.齐齐哈尔医学院，医学技术学院，黑龙江齐齐哈尔 161006

^1b.齐齐哈尔医学院，医药科学研究院，黑龙江齐齐哈尔 161006

通讯作者:

岳丽玲，教授，博士生导师 Tel: （0452）2663715 E-mail: yuell1025@126.com

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https://castjournals.cast.org.cn/joweb/zglcylxzz/CN/10.13699/j.cnki.1001-6821.2025.09.006

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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