PCK2 Promotes Malignant Phenotypes of Non-Small Cell Lung Cancer Cells via SLC38A2-Mediated Glutamine Uptake
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摘要:
目的 探讨在低糖环境下，磷酸烯醇式丙酮酸羧激酶2（phosphoenolpyruvate carboxykinase2，PCK2）调控谷氨酰胺（glutamine，Gln）转运的分子机制及其对非小细胞肺癌（non-small cell lung cancer，NSCLC）细胞恶性行为的影响。 方法 在人NSCLC细胞系A549上构建PCK2稳定敲低细胞系（KD组）及阴性对照（NC组）。所有体外实验均独立重复3次 （n = 3）。采用PRM蛋白质组学、Western blot和双荧光素酶报告基因实验检测PCK2对溶质载体家族38成员2（solute carrier family 38 member 2，SLC38A2）转录和蛋白表达的影响。在低糖条件 （ 1000 mg/L葡萄糖） 下，通过MTT法、划痕实验和流式细胞术分析细胞增殖、迁移和凋亡。最后设置Gln补充组（KD+Gln组）和SLC38A2过表达组（KD+SLC38A2组）进行功能挽救实验。结果 PCK2敲低后，SLC38A2启动子活性下降约60%（P < 0.0001 ），蛋白表达亦显著降低（P < 0.01）。低糖条件下，KD组细胞增殖受抑制，24 h迁移率（约20%）显著低于NC组（约55%，P <0.0001 ），凋亡率（约13%）显著高于NC组（约4.5%，P <0.0001 ）。补充Gln或过表达SLC38A2均能显著逆转上述现象，使迁移率恢复至约50%（P < 0.01 vs. KD组），凋亡率降至约5.5%（P <0.0001 vs. KD组）。结论 在低糖状态下，PCK2通过在转录水平上调控SLC38A2的表达，维持Gln摄取，从而促进NSCLC细胞的增殖、迁移和存活。PCK2-SLC38A2轴揭示了肿瘤代谢重编程的新机制，可能是一个潜在的NSCLC治疗靶点。 -
关键词:
- 非小细胞肺癌 /
- 磷酸烯醇式丙酮酸羧激酶2 /
- 溶质载体家族38成员2 /
- 谷氨酰胺转运 /
- 低糖环境
Abstract: To investigate the molecular mechanism by which phosphoenolpyruvate carboxykinase 2 （PCK2） regulates glutamine transport under low-glucose conditions and its impact on the malignant behaviors of non-small cell lung cancer （NSCLC） cells. Methods: Stable PCK2 knockdown （KD group） and negative control （NC group） cell lines （n = 3 for all experiments） were established in the human NSCLC A549 cell line.. The impact of PCK2 on the transcription and protein expression of solute carrier family 38 member 2 （SLC38A2） were assessed using PRM proteomics, Western blotting, and dual-luciferase reporter assays. Cell proliferation （MTT）, migration （wound healing）, and apoptosis （flow cytometry） were analyzed via MTT assay, scratch wound healing assay, and flow cytometry, respectively under low-glucose conditions （1000 mg/L）. Functional rescue experiments were performed by establishing a Gln supplementation group （KD+Gln group） and an SLC38A2 overexpression group （KD+SLC38A2 group）. Results: Upon PCK2 knockdown, SLC38A2 promoter activity decreased by approximately 60% （P <0.0001 ） and its protein expression was significantly reduced （P < 0.01）. Under low-glucose conditions, cell proliferation was inhibited in the KD group. The 24-hour migration rate （≈20%） was significantly lower than that in the NC group （≈55%, P <0.0001 ）, while the apoptosis rate （≈13%） was significantly higher （NC group ≈4.5%, P <0.0001 ）. Both glutamine supplementation and SLC38A2 overexpression significantly reversed these phenomena, restoring migration rates to approximately 50% （P < 0.01 vs. KD） and reducing apoptosis to about 5.5% （P <0.0001 vs. KD）. Conclusion: Under low-glucose stress, PCK2 maintains Gln uptake by regulating SLC38A2 expression at the transcriptional level, thereby promoting the proliferation, migration, and survival of NSCLC cells. The PCK2-SLC38A2 axis unveils a novel mechanism in tumor metabolic reprogramming and may represent a potential therapeutic target for NSCLC.-
Key words:
- Non-small cell lung cancer （NSCLC） /
- PCK2 /
- SLC38A2 /
- Glutamine transport /
- Low glucose
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图 1 PCK2对肺癌细胞中Gln转运蛋白表达的影响 （n = 3，$\bar x \pm s $）
A：使用qPCR验证对照（NC）组和PCK2敲低（KD）组中PCK2 mRNA表达水平；B：使用Western blot验证NC组和KD组中的相对PCK2蛋白表达水平；C：使用PRM靶向蛋白质组学定量Gln转运蛋白在NC组和KD组中的表达；D：PRM靶向蛋白质组学中Gln转运蛋白在NC组和KD组中的定量结果（KD/NC）；E：使用Western blot验证Gln转运蛋白在NC组和KD组中的蛋白水平，确认PRM结果；F：双荧光素酶实验验证PCK2对SLC38A2表达的影响。*P < 0.05； **P < 0.01； ***P < 0.001； ****P < 0.0001。
Figure 1. Effect of PCK2 on the expression of glutamine transporter proteins in lung cancer cells （n = 3，$\bar x \pm s $）
图 2 SLC38A1和SLC38A2表达水平与NSCLC患者总生存期的关系
A：SLC38A1在LUAD （Logrank P = 0.015） 和LUSC （Logrank P = 0.56） 患者中的生存曲线；B：SLC38A2在LUAD （Logrank P = 0.021） 和LUSC （Logrank P = 0.47） 患者中的生存曲线。
Figure 2. Relationship between expression levels of SLC38A1 and SLC38A2 and overall survival in NSCLC patients
图 3 PCK2调控及Gln供应恢复对低糖条件下肺癌细胞增殖、迁移和凋亡的影响 （n = 3，$\bar x \pm s $）
A：使用MTT法评估不同组别（NC、KD、KD+Gln）在5 d内的细胞增殖；B：使用创伤愈合实验评估细胞迁移率，在0 h、8 h和24 h进行观察。实验组：NC、KD、KD+Gln。左图为代表性图像，右图为定量结果；C：使用流式细胞术检测细胞凋亡率。实验组：NC、KD、KD+Gln。左图为代表性图，右图为定量结果。与NC组比较，**P < 0.01，****P < 0.0001，与KD组比较，##P < 0.01，####P < 0.0001。
Figure 3. Effects of PCK2 knockdown and glutamine supply restoration on proliferation，migration，and apoptosis of lung cancer cells under low-glucose conditions （n = 3，$\bar x \pm s $）
图 4 恢复Gln转运蛋白（SLC38A2）表达对低糖条件下PCK2敲低肺癌细胞增殖、迁移和凋亡的影响 （n = 3，$\bar x \pm s $）
A：使用qPCR验证NC、KD和KD+SLC38A2组的PCK2敲低效率；B：使用qPCR验证SLC38A2过表达效率；C：使用MTT法评估NC、KD和KD+SLC38A2组在5 d内的细胞增殖；D：使用创伤愈合实验评估细胞迁移率，在0 h、8 h和24 h时点进行观察。实验组：NC、KD、KD+SLC38A2。左图为代表性图像，右图为定量结果；E：使用流式细胞术检测细胞凋亡率。实验组：NC、KD、KD+SLC38A2。左图为代表性图，右图为定量结果。与NC组比较，**P < 0.0；***P < 0.001； ****P < 0.0001；与KD组比较，##P < 0.01； ###P < 0.001； ####P < 0.0001；ns为差异无统计学意义。
Figure 4. Effects of restoring glutamine transporter （SLC38A2） expression on proliferation，migration，and apoptosis of PCK2-knockdown lung cancer cells under low-glucose conditions （n = 3，$\bar x \pm s $）
表 1 qRT-PCR 引物表
Table 1. Primer sequences used for qRT-PCR
基因 引物 序列（5'→3'） PCK2 Forward （F） GTGGGGGATGATATTGCTTG PCK2 Reverse （R） TGGTCTCAGCCACATTGGTA SLC38A2 Forward （F） AGTTGCCTTTGGTGATCCAG SLC38A2 Reverse （R） CAGGACACGGAACCTGAAAT GAPDH Forward （F） CAGCCTCAAGATCATCAGCA GAPDH Reverse （R） ATGATGTTCTGGAGAGCCCC 
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表 2 UALCAN分析SLC38A1和SLC38A2与NSCLC的相关性
Table 2. UALCAN analysis of the associations between SLC38A1/SLC38A2 expression and NSCLC
基因 亚型 UALCAN
P 值高表达组中位
生存期 （月）高表达组
95%CI （月）低表达组
中位生存期 （月）低表达组
95%CI （月）GEPIA2 Logrank
P 值SLC38A2 LUAD 0.0053 130 120～160 180 150～210 0.021* LUSC 0.92 180 170～210 190 170～210 0.47 SLC38A1 LUAD 0.19 130 120～150 170 150～200 0.015* LUSC 0.44 200 150～220 210 170～230 0.56 *P < 0.05。
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