SERPINA3通过抑制铁死亡促进胰腺癌的恶性进展及吉西他滨耐药的机制研究

doi:10.19401/j.cnki.1007-3639.2025.06.004

摘要/Abstract

摘要：

背景与目的： 丝氨酸蛋白酶抑制剂（serine protease inhibitor，SERPIN）的家族成员可以通过调节肿瘤细胞的凋亡、侵袭、转移和血管生成等过程，影响肿瘤的发生、发展和预后，但其在胰腺癌中的作用机制尚不清楚。本研究旨探讨丝氨酸蛋白酶抑制剂A3蛋白（serine protease inhibitors A3，SERPINA3）高表达与胰腺癌细胞的增殖、凋亡、迁移及吉西他滨耐药之间的关系及机制。方法： 通过实时定量反转录聚合酶链反应（real-time quantitative reverse transcription polymerase chain reaction，qRT-PCR）分析正常胰腺导管上皮细胞系hTERT-HPNE与胰腺癌细胞系SW1990、Capan-1、PANC-1、AsPC-1中SERPINA3的表达水平；构建吉西他滨耐药胰腺癌细胞系PANC-1/R、AsPC-1/R，采用qRT-PCR实验、细胞计数试剂盒-8（cell counting kit-8，CCK-8）测定耐药及亲代敏感细胞系中SERPINA3表达水平及其对吉西他滨的药物敏感性差异。采用小干扰RNA（small interfering RNA，siRNA）构建SERPINA3敲低细胞系si-SERPINA，以siRNA negative control构建阴性对照组si-SERPINA#NC；利用丙二醛（malondialdehyde，MDA）、CCK-8、EdU细胞增殖实验、transwell迁移、matrigel侵袭、划痕和细胞凋亡实验，分别检测si-SERPINA组和si-SERPINA#NC组对吉西他滨耐药胰腺癌细胞的脂质过氧化水平、增殖、迁移、侵袭、修复能力和细胞凋亡的影响。结果： 与正常胰腺导管上皮细胞hTERT-HPNE相比，SERPINA3在多种胰腺癌细胞系中的表达水平显著升高（P<0.05）。相较于亲本细胞PANC-1和AsPC-1，其耐药衍生细胞PANC-1/R和AsPC-1/R细胞株中SERPINA3的mRNA表达和蛋白水平均显著升高（P<0.001）。当SERPINA3在PANC-1/R和AsPC-1/R细胞中被敲低后，细胞在不同浓度吉西他滨作用下的存活率下降，并且在MDA检测中显示出脂质过氧化水平增加（P<0.001）。此外，敲低SERPINA3的PANC-1/R和AsPC-1/R细胞系株的增殖率、迁移/侵袭细胞数量以及划痕实验的愈合率均显著降低（P<0.01），流式细胞术检测到凋亡细胞的数量增加（P<0.05）。这些结果表明，敲低SERPINA3能够抑制吉西他滨耐药胰腺癌细胞的增殖、迁移、侵袭和修复能力，并促进这些耐药细胞的凋亡。结论： SERPINA3在多种胰腺癌细胞中的呈高表达，SERPINA3高表达促进胰腺癌恶性进展和耐药，干扰SERPINA3表达可促进铁死亡并增强吉西他滨耐药胰腺癌细胞对药物的敏感性。

关键词: 胰腺癌, SERPINA3, 铁死亡, 吉西他滨, 耐药

Abstract:

Background and purpose: Members of the serine protease inhibitor (SERPIN) family can influence tumorigenesis, progression, and prognosis by modulating processes such as apoptosis, invasion, metastasis, and angiogenesis in tumor cells. However, their role in pancreatic cancer remains unclear. This study aimed to investigate the impact of high expression of serine protease inhibitor A3 (SERPINA3) on the proliferation, apoptosis, migration, and chemoresistance of pancreatic cancer cells and its mechanism. Methods: This study analyzed the SERPINA3 expression levels in the normal pancreatic ductal epithelial cell line hTERT-HPNE and pancreatic cancer cell lines SW1990, Capan-1, PANC-1, and ASPC-1 by real-time reverse transcription quantitative polymerase chain reaction (qRT-PCR). We established gemcitabine-resistant pancreatic cancer cell lines PANC-1/R and ASPC-1/R, and used qRT-PCR assay and cell counting kit-8 (CCK-8) to determine the SERPINA3 expression levels in the constructed resistant cell lines and their parental sensitive cell lines, as well as the differences in their chemosensitivity to gemcitabine. We constructed the SERPINA3-knockdown cell line si-SERPINA with siRNA, and the negative control group si-SERPINA#NC with siRNA negative control. We used MDA assay, CCK-8 assay, EdU cell proliferation assay, transwell migration assay, matrigel invasion assay, scratch assay, and apoptotic assay to respectively detect the lipid oxidation levels, proliferation, migration, invasion, wound-healing ability, and the influence on apoptosis of the gemcitabine-resistant pancreatic cancer cells in the si-SERPINA group and the si-SERPINA#NC group. Results: Compared with normal pancreatic ductal epithelial cells hTERT-HPNE, the expression level of SERPINA3 in various pancreatic cancer cell lines was significantly increased (P<0.05). mRNA and protein expression levels of SERPINA3 in PANC-1/R and ASPC-1/R were significantly increased compared with those in parent cells (P<0.001). When SERPINA3 was knocked down in PANC-1/R and ASPC-1/R cells, the survival rate of the cells under different concentrations of gemcitabine chemotherapy decreased, and MDA detected that the lipid oxidation level was increased (P<0.001). In addition, the proliferation rate of PANC-1/R and ASPC-1/R cell lines with SERPINA3 knockout, the number of migrating/invading cells and the healing rate of scratch test were significantly decreased (P<0.01), and flow cytometry demonstrated that the number of apoptotic cells was increased (P<0.05). These results suggest that SERPINA3 knockdown can inhibit the proliferation, migration, invasion and wound healing ability of gemcitabine-resistant pancreatic cancer cells, and promote the apoptosis of these resistant cells. Conclusion: SERPINA3 overexpression was found in various pancreatic cancer cells. SERPINA3 overexpression promoted malignant progression and chemotherapy resistance of pancreatic cancer, and interference with SERPINA3 expression promoted ferroptosis and enhanced chemotherapy sensitivity of gemcitabine-resistant pancreatic cancer cells.

Key words: Pancreatic cancer, SERPINA3, Ferroptosis, Gemcitabine, Drug resistance

中图分类号:

R735.9

和源, 郭俊成, 叶智斌, 王小虎, 李昊男, 黄敬彪. SERPINA3通过抑制铁死亡促进胰腺癌的恶性进展及吉西他滨耐药的机制研究[J]. 中国癌症杂志, 2025, 35(6): 555-562.

HE Yuan, GUO Juncheng, YE Zhibin, WANG Xiaohu, LI Haonan, HUANG Jingbiao. A research on the mechanism of SERPINA3 promoting malignant progression and gemcitabine resistance of pancreatic cancer by inhibiting ferroptosis[J]. China Oncology, 2025, 35(6): 555-562.

图/表 8

图1

图2

图3

图4

图5

图6

图7

图8

参考文献 16

[1]	BRAY F, LAVERSANNE M, SUNG H, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2024, 74(3): 229-263.
[2]	DIXON S J, OLZMANN J A. The cell biology of ferroptosis[J]. Nat Rev Mol Cell Biol, 2024, 25(6): 424-442.
[3]	CHEN Y M, JIANG Z L, ZHANG C X, et al. 5-Methylcytosine transferase NSUN2 drives NRF2-mediated ferroptosis resistance in non-small cell lung cancer[J]. J Biol Chem, 2024, 300(4): 106793.
[4]	ZHU M N, LAN Z H, PARK J, et al. Regulation of CNS pathology by Serpina3n/SERPINA3: The knowns and the puzzles[J]. Neuropathol Appl Neurobiol, 2024, 50(2): e12980.
[5]	MAWARIBUCHI S, SHIMOMURA O, ODA T, et al. rBC2LCN- reactive SERPINA3 is a glycobiomarker candidate for pancreatic ductal adenocarcinoma[J]. Glycobiology, 2023, 33(4): 342-352.
[6]	GUGENHEIM J, CROVETTO A, PETRUCCIANI N. Neoadjuvant therapy for pancreatic cancer[J]. Updates Surg, 2022, 74(1): 35-42.
[7]	WOOD L D, CANTO M I, JAFFEE E M, et al. Pancreatic cancer: pathogenesis, screening, diagnosis, and treatment[J]. Gastroenterology, 2022, 163(2): 386-402.e1.
[8]	LI Y, GUO L. The versatile role of Serpina3c in physiological and pathological processes: a review of recent s dies[J]. Front Endocrinol (Lausanne), 2023, 14: 1189007.
[9]	SOMAN A, ASHA NAIR S. Unfolding the cascade of SERPINA3: inflammation to cancer[J]. Biochim Biophys Acta Rev Cancer, 2022, 1877(5): 188760.
[10]	YANG H, GONG C, WU Y, et al. LncRNA SNHG1 facilitates colorectal cancer cells metastasis by recruiting HNRNPD protein to stabilize SERPINA3 mRNA[J]. Cancer Lett, 2024, 604: 217217.
[11]	LI G, SHE F F, LIAO C Y, et al. cNEK6 induces gemcitabine resistance by promoting glycolysis in pancreatic ductal adenocarcinoma via the SNRPA/PPA2c/mTORC1 axis[J]. Cell Death Dis, 2024, 15(10): 742.
[12]	DE LAAT V, TOPAL H, SPOTBEEN X, et al. Intrinsic temperature increase drives lipid metabolism towards ferroptosis evasion and chemotherapy resistance in pancreatic cancer[J]. Nat Commun, 2024, 15(1): 8540. doi: 10.1038/s41467-024-52978-z pmid: 39358362
[13]	LI Y, RAN Q, DUAN Q, et al. 7-dehydrocholesterol dictates ferroptosis sensitivity[J]. Nature, 2024, 626(7998): 411-8.
[14]	KIM M J, KIM H S, KANG H W, et al. SLC38A5 modulates ferroptosis to overcome gemcitabine resistance in pancreatic cancer[J]. Cells, 2023, 12(20).
[15]	SUN S, SHEN J, JIANG J, et al. Targeting ferroptosis opens new avenues for the development of novel therapeutics[J]. Signal Transduct Target Ther, 2023, 8(1): 372.
[16]	SHI J F, LIU Y, WANG Y, et al. Targeting ferroptosis, a novel programmed cell death, for the potential of alcohol-related liver disease therapy[J]. Front Pharmacol, 2023, 14: 1194343.