DTX2促进奥沙利铂耐药的结直肠癌细胞增殖、侵袭和上皮间质转化

doi:10.12122/j.issn.1673-4254.2025.04.18

摘要/Abstract

摘要：

目的探讨DTX2对奥沙利铂耐药的结直肠癌（CRC/OXA）细胞的影响及作用机制。方法利用CCK8检测奥沙利铂（OXA）对CRC细胞的抑制率，构建CRC/OXA细胞系，检测CRC/OXA细胞中DTX2的表达水平，利用基因工具干预CRC/OXA细胞，分为未转染组（con）、敲低组（DTX2-shRNA）及共转染组（DTX2-shRNA+ pcDNA-Notch2）。采用平板克隆、划痕和Transwell侵袭实验检测改变DTX2的表达对CRC/OXA细胞增值、迁移侵袭能力的影响，并通过Western blotting检测各组中Notch2、NICD、E-cadherin、N-cadherin及Vimentin蛋白的表达水平。利用SW620/OXA细胞同样分组行裸鼠移植瘤实验，体内验证对裸鼠成瘤及蛋白的影响。结果 OXA对CRC细胞有明显抑制作用，SW620和LoVo细胞IC₅₀分别为6.00和8.00 μmol/L，成功构建CRC/OXA细胞系，CRC/OXA细胞中DTX2表达量明显升高（P<0.01）。DTX2-shRNA组中CRC/OXA细胞明显抑制增值、迁移侵袭能力（P<0.05），DTX2-shRNA+pcDNA-Notch2组可逆转增强CRC/OXA细胞增值、迁移侵袭的能力（P<0.05）。Notch2、NICD及Vimentin蛋白平均表达水平，在DTX2-shRNA组中明显降低，而DTX2-shRNA+pcDNA-Notch2组明显升高（P<0.01）；E-cadherin蛋白表达水平在以上两组中的趋势相反且有明显差异（P<0.01）。体内实验显示DTX2可明显促进SW620/OXA细胞移植瘤的生长及对应蛋白的变化（P<0.05）。结论 DTX2通过Notch2信号通路促进 CRC/OXA细胞增值、迁移侵袭及上皮间质转化，DTX2可能作为提高OXA疗效的分子标志物。

关键词: deltex-2, 结直肠癌, Notch2信号通路, 上皮间质转化, 奥沙利铂，耐药

Abstract:

Objective To investigate the role of DTX2 in regulating biological behaviors of oxaliplatin-resistant colorectal cancer cells (CRC/OXA cells). Methods CCK8 assay was used to determine the inhibition rate of oxaliplatin-treated CRC cells. A CRC/OXA cell line was constructed, in which DTX2 expression level was detected. The cells were transfected with a DTX2-shRNA plasmid or co-transfected with DTX2-shRNA and pcDNA-Notch2, and the changes in cell proliferation, migration and invasion ability were evaluated using plate cloning assay, scratch assay and Transwell invasion assay. The expression levels of Notch2, NICD and epithelial-mesenchymal transition (EMT) proteins of the transfected cells were detected with Western blotting. In a nude mouse model bearing SW620/OXA cell xenografts, the effects of DTX2 knockdown and Notch2 overexpression in the implanted cells on tumor growth and protein expressions were tested. Results The IC₅₀ of oxaliplatin was 6.00 μmol/L in SW620 cells and 8.00 μmol/L in LoVo cells. CRC/OXA cells showed a significantly increased expression of DTX2. DTX2 knockdown in CRC/OXA cells significantly inhibited cell proliferation, migration and invasion, and these effects were reversed by co-transfection of the cells with pcDNA-Notch2. DTX2 knockdown significantly reduced the expression levels of Notch2, NICD and vimentin proteins and increased E-cadherin expression in CRC/OXA cells, and co-transfection with pcDNA-Notch2 potently attenuated the changes in these proteins. In the tumor-bearing mice, DTX2 overexpression obviously promoted the growth of SW620/OXA cell xenograft, enhanced the protein expressions of Notch2, NICD and vimentin, and lowered the expression of E-cadherin. Conclusion High expression of DTX2 promotes proliferation, migration, invasion and EMT of CRC/OXA cells through the Notch2 signaling pathway, suggesting the potential of DTX2 as a target to improve the efficacy of oxaliplatin.

Key words: deltex-2, colorectal cancer, Nohch2 signaling pathway, epithelial-mesenchymal transition, oxaliplatin resistance

马振南, 刘福全, 赵雪峰, 张晓微. DTX2促进奥沙利铂耐药的结直肠癌细胞增殖、侵袭和上皮间质转化[J]. 南方医科大学学报, 2025, 45(4): 829-836.

Zhennan MA, Fuquan LIU, Xuefeng ZHAO, Xiaowei ZHANG. High expression of DTX2 promotes proliferation, invasion and epithelial-mesenchymal transition of oxaliplatin-resistant colorectal cancer cells[J]. Journal of Southern Medical University, 2025, 45(4): 829-836.

图/表 8

图1 CRC细胞的IC50值

Fig.1 IC50 of oxaliplatin in SW620 cells (6.00 μmol/L) and LoVo cells (8.00 μmol/L).

图2 CRC/OXA细胞中DTX2 mRNA和蛋白的表达水平

Fig 2 Western blotting for detecting protein expressions of DTX2 (A) and its relative protein (B) and mRNA (C) expression levels in CRC and CRC/OXA cells. SW620/OXA: Oxaliplatin-resistant SW620 cells; LoVo /OXA: Oxaliplatin-resistant LoVo cell. *P<0.01.

图3 CRC/OXA细胞共转染DTX2-shRNA和pcDNA-Notch2后对其增殖能力的影响

Fig.3 Plate cloning assays showing the proliferation capacity of CRC/OXA cells after transfection with DTX2-shRNA and DTX2-shRNA+pcDNA-Notch2 (A) and the mean number of colonies formed (B). *P<0.05.

图4 CRC/OXA细胞共转染DTX2-shRNA和pcDNA-Notch2后对其迁移能力的影响

Fig.4 Scratch assay for assessing changes of migration ability of SW620/OXA (A, B) and LoVo/OXA (C, D) cells after transfection with DTX2-shRNA and DTX2-shRNA+pcDNA-Notch2. *P<0.05.

图5 CRC/OXA细胞共转染DTX2-shRNA和pcDNA-Notch2后对其侵袭能力的影响

Fig.5 Transwell invasion assay for assessing changes of migration ability of SW620/OXA and LoVo/OXA cells after transfection with DTX2-shRNA and DTX2-shRNA+pcDNA-Notch2. A: Microscopic observation of the cells (Original magnification: ×100). B: Average number of invasive cells in different groups. *P<0.01.

图6 CRC/OXA细胞共转染DTX2-shRNA和pcDNA-Notch2后Notch2信号通路及EMT中相关蛋白的变化

Fig.6 Changes of protein expressions in the Notch2 Signal pathway and EMT in SW620 and LoVo cells co-transfected with DTX2-shRNA and DTX2-shRNA+pcDNA-Notch2. A, C: Western blots of the proteins in SW620/OXA and LoVo/OXA cells. B, D: Quantitative analysis of the protein expressions. GAPDH was used as the internal control. *P<0.01.

图7 SW620/OXA细胞干预后对裸鼠移植瘤的影响

Fig.7 Growth of SW620/OXA cell xenografts transfected with DTX2-shRNA or DTX2-shRNA+pcDNA-Notch2 in nude mice. A: Observation of the tumor-bearing mice and the dissected tumors. B: Changes in the volume of the xenografts over time. C: Xenograft weight measurement. *P<0.05.

图8 SW620/OXA细胞裸鼠成瘤组织中蛋白的影响

Fig.8 Protein expressions in SW620/OXA cell xenografts transfected with DTX2-shRNA or DTX2-shRNA+pcDNA-Notch2. A: Western blotting of the proteins in SW620/OXA cell xenografts. B: Quantitative analysis of the protein expressions. GAPDH was used as the internal control. *P<0.01.

参考文献 34

1	Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2021, 71(3): 209-49.
2	Guo JF, Yu Z, Das M, et al. Nano codelivery of oxaliplatin and folinic acid achieves synergistic chemo-immunotherapy with 5-fluorouracil for colorectal cancer and liver metastasis[J]. ACS Nano, 2020, 14(4): 5075-89.
3	Rasmussen MH, Lyskjær I, Jersie-Christensen RR, et al. miR-625-3p regulates oxaliplatin resistance by targeting MAP2K6-p38 signalling in human colorectal adenocarcinoma cells[J]. Nat Commun, 2016, 7: 12436.
4	Zacharakis G, Almasoud A, Arahmaner O, et al. A 5-year evaluation of early-and late-onset sporadic colorectal cancer screening in central Saudi Arabia[J]. Saudi J Gastroenterol, 2023, 29(2): 95-101.
5	Shi YX, Niu Y, Yuan YC, et al. PRMT3-mediated arginine methylation of IGF2BP1 promotes oxaliplatin resistance in liver cancer[J]. Nat Commun, 2023, 14(1): 1932.
6	Wu SZ, Shen SH, Lu F, et al. Bromodomain containing 4 transcriptionally activated Deltex E3 ubiquitin ligase 2 contributes to glioma progression and predicts an unfavorable prognosis[J]. Ann Transl Med, 2022, 10(6): 313.
7	Li R, Chen Y, Yang B, et al. DTX2 promotes glioma development via regulation of HLTF[J]. Biol Direct, 2024, 19(1): 2.
8	黄小强. 基于生信分析DTX2相关基因在肝癌中的表达及临床价值[D]. 广州: 南方医科大学, 2024.
9	Liu Z, Liu C, Fan CH, et al. E3 ubiquitin ligase DTX2 fosters ferroptosis resistance via suppressing NCOA4-mediated ferritinophagy in non-small cell lung cancer[J]. Drug Resist Updat, 2024, 77: 101154.
10	Song M, Kuerban M, Zhao L, et al. Inhibition of RFX6 suppresses the invasive ability of tumor cells through the Notch pathway and affects tumor immunity in hepatocellular carcinoma[J]. Front Oncol, 2021, 11: 801222.
11	马振南, 许广大, 刘福全, 等. 结直肠癌组织中DTX2分子的表达及临床意义[J]. 中国普外基础与临床杂志, 2021, 28(7): 861-6.
12	马振南, 赵雪峰, 张晓微, 等. DTX2通过Notch2/Akt轴促进结直肠癌细胞的迁移和侵袭[J]. 南方医科大学学报, 2023, 43(3): 340-8.
13	De Mattia E, Dreussi E, Montico M, et al. A clinical-genetic score to identify surgically resected colorectal cancer patients benefiting from an adjuvant fluoropyrimidine-based therapy[J]. Front Pharmacol, 2018, 9: 1101.
14	邓金海, 潘腾, 周广林, 等. 高表达分泌颗粒蛋白Ⅱ增加结直肠癌细胞对奥沙利铂的耐药性[J]. 南方医科大学学报, 2023, 43(10): 1657-64.
15	Mayer RJ. Flashback foreword: oxaliplatin plus LV5FU2 in colorectal cancer[J]. J Clin Oncol, 2023, 41(33): 5077-8.
16	Cai M, Hu WL, Huang CJ, et al. lncRNA MCF2L-AS1/miR-105/IL-1β axis regulates colorectal cancer cell oxaliplatin resistance[J]. Cancer Manag Res, 2021, 13: 8685-94.
17	Mora Y, Reyes ME, Zanella L, et al. Resistance to platinum-based cancer drugs: a special focus on epigenetic mechanisms[J]. Pharmacogenomics, 2021, 22(12): 777-90.
18	Kosugi C, Koda K, Ishibashi K, et al. Safety of mFOLFOX6/XELOX as adjuvant chemotherapy after curative resection of stage III colon cancer: phase II clinical study (The FACOS study)[J]. Int J Colorectal Dis, 2018, 33(6): 809-17.
19	Li HR, Yang BB. Friend or foe: the role of microRNA in chemo-therapy resistance[J]. Acta Pharmacol Sin, 2013, 34(7): 870-9.
20	Yu ZL, Deng P, Chen YF, et al. Inhibition of the PLK1-coupled cell cycle machinery overcomes resistance to oxaliplatin in colorectal cancer[J]. Adv Sci, 2021, 8(23): e2100759.
21	Zhang CY, Xu C, Gao XY, et al. Platinum-based drugs for cancer therapy and anti-tumor strategies[J]. Theranostics, 2022, 12(5): 2115-32.
22	Hölzel M, Bovier A, Tüting T. Plasticity of tumour and immune cells: a source of heterogeneity and a cause for therapy resistance [J]? Nat Rev Cancer, 2013, 13(5): 365-76.
23	McMillin DW, Negri JM, Mitsiades CS. The role of tumour-stromal interactions in modifying drug response: challenges and opportunities[J]. Nat Rev Drug Discov, 2013, 12(3): 217-28.
24	Wang Q, Chen X, Jiang YH, et al. Elevating H3K27me3 level sensitizes colorectal cancer to oxaliplatin[J]. J Mol Cell Biol, 2020, 12(2): 125-37.
25	Dhanyamraju PK. Drug resistance mechanisms in cancers: execution of pro-survival strategies[J]. J Biomed Res, 2024, 38(2): 95-121.
26	Chen G, Gong T, Wang Z, et al. Colorectal cancer organoid models uncover oxaliplatin- resistant mechanisms at single cell resolution [J]. Cell Oncol (Dordr), 2022, 45(6):1155-67.
27	Aliabadi F, Sohrabi B, Mostafavi E, et al. Ubiquitin-proteasome system and the role of its inhibitors in cancer therapy[J]. Open Biol, 2021, 11(4): 200390.
28	Han DW, Wang LJ, Jiang S, et al. The ubiquitin-proteasome system in breast cancer[J]. Trends Mol Med, 2023, 29(8): 599-621.
29	Park J, Cho J, Song EJ. Ubiquitin-proteasome system (UPS) as a target for anticancer treatment[J]. Arch Pharm Res, 2020, 43(11): 1144-61.
30	李向阳. 甲状腺乳头状癌中DTX2的表达与临床意义[D]. 沈阳: 中国医科大学, 2018.
31	Cui YH, Wei JB, Fan H, et al. Targeting DTX2/UFD1-mediated FTO degradation to regulate antitumor immunity[J]. Proc Natl Acad Sci USA, 2024, 121(51): e2407910121.
32	Maki K, Sasaki K, Sugita F, et al. Acute myeloid leukemia with t(7;21)(q11.2;q22) expresses a novel, reversed-sequence RUNX1-DTX2 Chimera[J]. Int J Hematol, 2012, 96(2): 268-73.
33	Yonezawa T, Takahashi H, Hao YY, et al. The E3 ligase DTX2 inhibits RUNX1 function by binding its C terminus and prevents the growth of RUNX1-dependent leukemia cells[J]. FEBS J, 2023, 290(21): 5141-57.
34	Zhou BH, Lin WL, Long YL, et al. Notch signaling pathway: architecture, disease, and therapeutics[J]. Signal Transduct Target Ther, 2022, 7(1): 95.

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