NPC2蛋白通过MEK/ERK和JNK信号通路在胃癌进展中的研究

doi:10.3877/cma.j.issn.2095-2015.2026.05.007

目的

探讨NPC2（Niemann-Pick C2）蛋白在胃癌细胞中的作用及MEK/ERK和JNK信号通路调控机制。

方法

检测正常人体细胞和胃癌细胞系中NPC2表达；评估细胞增殖、克隆、迁移及侵袭能力，并检测通路磷酸化水平。

结果

胃癌细胞中的NPC2表达明显高于正常上皮细胞，且AGS细胞中NPC2表达最高（P<0.01）。NPC2的敲除显著抑制了细胞的增殖、克隆形成、迁移及侵袭能力（P<0.05）。NPC2的敲除导致MEK/ERK和JNK通路的磷酸化水平显著降低（P<0.05）。过表达NPC2能够促进AGS细胞的增殖、克隆形成、迁移和侵袭能力（P<0.05），而MEK/ERK和JNK通路抑制剂E6201则抑制了这些生物学过程。

结论

NPC2通过激活MEK/ERK和JNK信号通路促进胃癌进展，可能是潜在治疗靶点。

关键词: 胃癌, NPC2蛋白, 细胞增殖, 迁移和侵袭

Objective

To investigate the role of NPC2 (Niemann-Pick C2) protein in gastric cancer cells and its regulatory mechanisms through the MEK/ERK and JNK signaling pathways.

Methods

The expression levels of NPC2 in normal human cells and gastric carcinoma cell lines were detected. The cell proliferation, cloning, migration and invasion abilities were evaluated, and the phosphorylation levels of the pathways were detected.

Results

NPC2 expression in gastric cancer cells was significantly higher than that in normal epithelial cells, with the highest expression observed in AGS cells (P<0.01). Knockdown of NPC2 significantly inhibited cell proliferation, colony formation, migration, and invasion abilities (P<0.05). NPC2 knockdown also resulted in a significant decrease in the phosphorylation levels of the MEK/ERK and JNK pathways (P<0.05). Overexpression of NPC2 promoted AGS cell proliferation, colony formation, migration, and invasion (P<0.05), while the MEK/ERK and JNK pathway inhibitor E6201 inhibited these biological processes.

Conclusion

NPC2 promotes the progression of gastric cancer by activating the MEK/ERK and JNK signaling pathways, and may represent a potential therapeutic target.

Key words: Gastric cancer, NPC2 protein, Cell proliferation, Migration and invasion

表1 qPCR引物序列

基因名称	引物序列
NPC2	Forward(F): 5'-TGTGGCTCAGTGGCTTAGG-3'
NPC2	Reverse(R): 5'-CCAGGAAGGGATTTCACACA-3'
MEK	F: 5'-TTGGTGAGCGTGTGGTGTCTG-3'
MEK	R:5'-CAGGTGGGTGTGGGTGTGAAG-3'
ERK	F:5'-GCTCTGTCTCCAGGCTGAAGTG
ERK	R:5'-GCTGAGGAAGAAGAATCGCTTGAAC
JNK	F:5'-CTGGACCAAGCGGACCTAATAGAC
JNK	R:5'-AGTGTGATGTGGTGCTGAGAAGAG
β-actin	F: 5'-ACCCCAAAGCCAACAGA-3'
β-actin	R: 5'-CCAGAGTCCATCACAATACC-3'

表1 qPCR引物序列

基因名称	引物序列
NPC2	Forward(F): 5'-TGTGGCTCAGTGGCTTAGG-3'
NPC2	Reverse(R): 5'-CCAGGAAGGGATTTCACACA-3'
MEK	F: 5'-TTGGTGAGCGTGTGGTGTCTG-3'
MEK	R:5'-CAGGTGGGTGTGGGTGTGAAG-3'
ERK	F:5'-GCTCTGTCTCCAGGCTGAAGTG
ERK	R:5'-GCTGAGGAAGAAGAATCGCTTGAAC
JNK	F:5'-CTGGACCAAGCGGACCTAATAGAC
JNK	R:5'-AGTGTGATGTGGTGCTGAGAAGAG
β-actin	F: 5'-ACCCCAAAGCCAACAGA-3'
β-actin	R: 5'-CCAGAGTCCATCACAATACC-3'

图1 NPC2的表达及转染效率的鉴定　1A：RT-qPCR检测各种细胞系中NPC2的表达水平；1B：明场（Bright field）和荧光显微镜下细胞密度和细胞GFP荧光表达情况；1C：WB检测各组AGS细胞中NPC2蛋白的表达水平；1D：WB定量；1E：qPCR检测各组AGS细胞中NPC2 mRNA的表达水平（与Control比较，^*P<0.05，^**P<0.01，^****P<0.0001）

图2 NPC2对细胞增殖、迁移和侵袭的影响　2A：加入不同浓度SY-LB-35细胞的活力；2B：各组细胞1~7 d的增殖曲线；2C：各组细胞的克隆形成情况；2D：各组细胞克隆形成统计情况；2E：划痕实验；2F：各组细胞的迁移情况情况；2G：Transwell侵袭实验，bar=200 μm；2H：各组细胞侵袭能力情况（与NC比较，^*P<0.05，^**P<0.01；与NC＋SY比较，^###P<0.001）

图3 NPC2和MEK/ERK信号通路激动剂对AGS细胞MEK/ERK信号通路蛋白表达的影响　3A：WB检测各组细胞NPC2、MEK/ERK和JNK的蛋白表达情况；3B：RT-qPCR检测NPC2的水平；3C：qRT-PCR检测各组细胞MEK/ERK和JNK表达水平的结果分析；3D：WB检测NPC2的表达水平统计情况；3E：各组细胞MEK1/2、ERK1/2和JNK1/2/3的相对磷酸化水平（与NC比较，^*P<0.05，^**P<0.01，^***P<0.001；与NC＋SY比较，^#P<0.05，^##P<0.01，^###P<0.001）

图4 过表达NPC2对细胞增殖、迁移和侵袭的影响　4A：明场（Bright field）和荧光显微镜下细胞密度和细胞GFP荧光表达情况，bar=500 μm；4B：RT-qPCR检测各组AGS细胞中NPC2蛋白的表达水平；4C：WB检测各组AGS细胞中NPC2 mRNA的表达水平；4D：不同浓度E6201对细胞活力的影响；4E：细胞增殖曲线；4F~4G：克隆形成；4H~4I：细胞迁移实验；4J~4K：细胞侵袭实验，bar=200 μm（与NC比较，^*P<0.05，^**P<0.01，^***P<0.001；与NC＋E6201比较，^###P<0.001，^####P<0.0001）

图5 过表达NPC2和MEK/ERK信号通路激动剂对AGS细胞MEK/ERK信号通路蛋白表达的影响　5A：WB检测各组细胞NPC2、MEK/ERK和JNK的蛋白表达情况；5B：RT-qPCR检测NPC2的水平；5C：RT-qPCR检测各组细胞MEK/ERK和JNK表达水平的结果分析；5D：WB检测NPC2的表达水平统计情况；5E：各组细胞MEK1/2、ERK1/2和JNK1/2/3的相对磷酸化水平（与NC比较，^*P<0.05，^**P<0.01；与NC＋E6201比较，^#P<0.05，^##P<0.01）

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Role of NPC2 protein in promoting gastric cancer progression through the MEK/ERK and JNK signaling pathways

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Role of NPC2 protein in promoting gastric cancer progression through the MEK/ERK and JNK signaling pathways