内质网蛋白驻留受体2蛋白对胰腺癌恶性生长的影响及机制研究

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摘要目的：探讨赖氨酸天冬氨酸谷氨酸亮氨酸（KDEL）内质网蛋白驻留受体2（endoplasmic reticulum protein retention receptor 2,KDELR2）对胰腺癌细胞恶性生长的影响，并初步探究相关作用分子机制。方法：通过癌症基因组学数据分析平台（UCSC XENA）分析KDELR2 mRNA在泛癌和人胰腺癌组织中的表达水平；通过癌症基因组图谱（The Cancer Genome Atlas,TCGA）分析其表达水平与胰腺癌患者临床病理特征及生存预后的关系；采用基因本体功能富集分析（Gene Ontology，GO）、京都基因和基因组数据库富集分析（Kyoto Encyclopedia of Genes and Genomes，KEGG）探索KDELR2相关差异表达基因参与调节的信号通路；通过ssGSEA算法分析KDELR2免疫浸润情况。应用实时荧光定量PCR(qRTPCR)和蛋白质印迹实验检测不同胰腺癌细胞系(BXPC-3、MIA PaCa-2、PANC-1、PaTu 8988-T)以及正常人胰腺导管上皮细胞(HPNE)中KDELR2的mRNA和蛋白表达水平，并检测人胰腺癌组织及对应癌旁组织中KDELR2的蛋白表达水平。通过慢病毒转染构建KDELR2过表达和敲减的稳转细胞株(PaTu 8988-T和PANC1)及其对照空载体细胞株(“KDELR2-OE”和“Vec”、“shKDELR2-S1/S2”和“shNC”)，利用CCK8、EdU、Transwell、细胞克隆形成实验观察KDELR2过表达及敲低后稳转细胞株的生物学行为变化；使用JC1、MitoSOX和DCFHDA染色实验以及检测ATP水平观察PaTu 8988T和PANC1细胞KDELR2敲低后的线粒体功能。通过构建裸鼠皮下荷瘤模型观察KDELR2敲低对胰腺癌PANC1细胞在体生长的影响。结果： KDELR2在人胰腺癌组织及细胞中显著高表达，其高表达水平与胰腺癌患者的病理特征及不良预后密切相关。GO功能和KEGG富集分析表明，KDELR2可能通过调控细胞能量代谢调节胰腺癌细胞生长。敲低KDELR2显著抑制胰腺癌细胞的增殖和迁移能力，过表达KDELR2显著促进胰腺癌细胞的增殖和迁移能力；KDELR2敲低后，胰腺癌细胞线粒体膜电位明显下降，活性氧水平明显升高，ATP水平明显降低（P均<0.05）。裸鼠皮下荷瘤模型证实KDELR2敲低显著抑制胰腺癌细胞的在体生长。结论： KDELR2在胰腺癌中显著高表达且与患者不良预后密切相关，KDELR2可能参与调控线粒体的功能影响癌细胞能量代谢。

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	王书航1
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Abstract： Objective： To investigate the effect of KDELR2 on the malignant growth of pancreatic cancer cells, and its underlying molecular mechanisms. Methods： The expression of KDELR2 mRNA in pan-cancer and human pancreatic cancer tissues was analyzed by UCSC XENA database, and the association of its expression with clinicopathological characteristics and prognosis of pancreatic cancer patients was analyzed based on TCGA database. GO and KEGG analyses were used to explore the potential enriched signaling pathways of KDELR2related differentially expressed genes (DEGs). qRT-PCR and Western blotting were respectively applied to detect the KDELR2 mRNA and protein expression levels in different pancreatic cancer cell lines (BXPC-3, MIA PaCa-2, PANC-1, and PaTu 8988-T), as well as normal HPNE, and the KDELR2 protein level of human pancreatic cancer tissues and paracancerous tissues were also tested. Stable pancreatic cancer cells (PaTu 8988-T and PANC-1) containing the lentiviral particles encoding KDELR2 cDNA or KDELR2 shRNA and their scramble control lentiviral particles (“KDELR2-OE” and “Vec”, “shKDELR2S1/S2”and “shNC”) were formed. In addition, CCK-8, EdU staining, Transwell, and cell colony formation experiments were applied to observe the functional change of those stable cell lines. The mitochondrial function of PaTu 8988-T and PANC-1 cells after KDELR2 knockdown was observed by JC-1, MitoSOX and DCFH-DA staining assays and ATP level detection assays respectively. Furthermore, the effect of KDELR2 shRNA on the growth of PANC-1 cells in vivo was explored by constructing a subcutaneous xenograft tumor model in nude mice. Results： KDELR2 was overexpressed in human pancreatic cancer tissues and cells, and its overexpression was positively correlated with higher tumor clinicopathological stages, indicating a poor prognosis of pancreatic cancer patients. GO function and KEGG enrichment analysis suggested that KDELR2 may regulate the growth of pancreatic cancer cells by regulating cellular energy metabolism. After silencing of KDELR2, the growth, proliferation and migration of pancreatic cancer cells were significantly inhibited. Ectopic overexpression of KDELR2 significantly promoted the proliferation and migration of pancreatic cancer cells. After KDELR2 knockdown, pancreatic cancer cells showed a significant decrease in mitochondrial membrane potential and ATP levels, a significant increase in reactive oxygen species levels (all P<0.05). A subcutaneous xenograft model in nude mice demonstrated that KDELR2 knockdown significantly inhibited the growth of PANC-1 cells in vivo. Conclusion： KDELR2 is overexpressed in pancreatic cancer tissues and correlated with poor prognosis of pancreatic cancer patients, and KDELR2 may be involved in regulating mitochondrial function and energy metabolism in cancer cells.

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收稿日期: 2023-11-30

基金资助: 国家自然科学基金资助项目（82072712）

通讯作者: 吴晓阳（通讯作者），硕士生导师，副教授，E-mail： wxy3339@163.com

作者简介: 王书航(1998—)，男，硕士研究生

引用本文:

王书航1，吴晓阳2. 内质网蛋白驻留受体2蛋白对胰腺癌恶性生长的影响及机制研究[J]. 江苏大学学报:医学版, 2024, 34(02): 118-131. WANG Shuhang1, WU Xiaoyang2. Effect of KDEL endoplasmic reticulum protein retension receptor 2 on the malignant growth of pancreatic cancer and potential mechanisms. Journal of Jiangsu University(Medicine Edition), 2024, 34(02): 118-131.

链接本文:

http://zzs.ujs.edu.cn/xbyxb/CN/ 或 http://zzs.ujs.edu.cn/xbyxb/CN/Y2024/V34/I02/118

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