基于光学表面波技术探究具核梭杆菌介导的自噬与细胞折射率的关系

doi:10.6040/j.issn.1671-7554.0.2024.0652

摘要/Abstract

摘要： 目的拟利用光学表面波传感技术,探索具核梭杆菌(fusobacterium nucleatum, Fn)介导的自噬水平与细胞折射率变化的关系,初步探究光学表面波技术对癌细胞自噬水平检测的潜力。方法运用CCK-8技术检测Fn对结直肠癌细胞增殖活力的影响,蛋白免疫印迹法及自噬双标荧光法检测Fn对自噬水平的影响,同时采用光学表面波技术检测不同Fn感染状态下细胞折射率的变化,观察其与自噬水平的相关性。结果当感染复数为100,感染时间为4 h时,自噬起始蛋白苄氯素1(Beclin-1)、微管相关蛋白 1A/1B-轻链 3(microtubule-associated protein 1A/1B light chain 3,LC3)蛋白表达水平升高(均P<0.05),螯合体1(sequestome, P62/SQSTM1)蛋白表达水平降低(P<0.01),自噬水平升高;同时光学检测结果显示,HCT116、HT29细胞的折射率亦随自噬水平的增高而升高(均P<0.01)。进一步研究表明,细胞折射率的变化主要在于细胞内自噬小体水平的变化,且不受细胞增殖及胞外物质等因素影响。结论 Fn介导的自噬水平与细胞折射率具有相关性,可实时反映Fn介导的自噬水平的变化,表明光学表面波技术可作为一种快速、直观且不影响细胞活性的检测自噬水平的技术。

关键词: 具核梭杆菌, 结直肠癌, 自噬, 光学表面波技术, 细胞折射率

Abstract: Objective To explore the relationship between autophagy levels mediated by Fusobacterium nucleatum(Fn)and changes in cellular refractive index using optical surface wave sensing technology and to preliminarily investigates the potential of optical surface wave technology for detecting autophagy levels in cancer cells. Methods CCK-8 was used to assess the impact of Fn on the proliferation activity of colorectal cancer cells. The effects of Fn on autophagy levels were examined using both Western blotting and dual-labeling fluorescence microscopy techniques. Additionally, optical surface wave technology was employed to monitor changes in cellular refractive index under different Fn infection states, and to observe their correlation with autophagy levels. Results When multiplicity of infection was 100 and the infection time was 4 hours, the protein expression levels of Beclin-1 and LC3 were increased(P<0.05), while the expression level of P62 was decreased(P<0.01), indicating elevated autophagy levels. Concurrently, optical surface wave showed that the refractive indices of HCT116 and HT29 cells also increased with the elevation of autophagy levels(P<0.01). Further investigation revealed that changes in cellular refractive index were primarily due to variations in autophagosome levels within the cells, and were not influenced by factors such as cell proliferation or extracellular substances. Conclusion The autophagy levels mediated by Fn correlate with cellular refractive index, indicating that optical surface wave sensing technology can dynamically reflect changes in Fn-mediated autophagy levels. This suggests that optical surface wave technology could serve as a rapid, intuitive, and non-invasive method to assess autophagy levels without affecting cellular viability.

Key words: Fusobacterium nucleatum, Colorectal cancer, Autophagy, Optical surface wave technique, Cell refractive index

中图分类号:

于丽,王弋嘉,杨勇,刘学焕,万雪花,包翠萍,苗蓓亮,李斯琪,李静,刘筠. 基于光学表面波技术探究具核梭杆菌介导的自噬与细胞折射率的关系[J]. 山东大学学报 (医学版), 2024, 62(10): 87-97.

YU Li, WANG Yijia, YANG Yong, LIU Xuehuan, WAN Xuehua, BAO Cuiping, MIAO Beiliang, LI Siqi, LI Jing, LIU Jun. Relationship between fusobacterium nucleatum mediated autophagy and cell refractive index based on optical surface wave[J]. Journal of Shandong University (Health Sciences), 2024, 62(10): 87-97.

参考文献

[1] Dekker E, Tanis PJ, Vleugels JLA, et al. Colorectal cancer[J]. Lancet, 2019, 394(10207): 1467-1480.
[2] Mahdavi M, Laforest-Lapointe I, Massé E. Preventing colorectal cancer through prebiotics[J]. Microorganisms, 2021, 9(6): 1325. doi:10.3390/microorganisms9061325.
[3] 上海市抗癌协会大肠癌专业委员会. 结直肠癌早筛、早诊、早治上海方案(2023年版)[J]. 中国癌症杂志, 2024, 34(1): 13-66. Colorectal Cancer Special Committee of Shanghai Anti-Cancer Association. Shanghai plan for early screening, diagnosis and treatment of colorectal cancer(2023 edition)[J]. China Oncology, 2024, 34(1): 13-66.
[4] 苏雅丽, 曹秋华, 高兴华. 结直肠癌的风险因素研究进展[J]. 药学研究, 2023, 42(8): 594-598, 640. SU Yali, CAO Qiuhua, GAO Xinghua. Research progress on risk factors for colorectal cancer[J]. Journal of Pharmaceutical Research, 2023, 42(8): 594-598, 640.
[5] Lin CZ, Cai XL, Zhang J, et al. Role of gut microbiota in the development and treatment of colorectal cancer[J]. Digestion, 2019, 100(1): 72-78.
[6] Zepeda-Rivera M, Minot SS, Bouzek H, et al. A distinct Fusobacterium nucleatum clade dominates the colorectal cancer niche[J]. Nature, 2024, 628(8007): 424-432.
[7] Tahara T, Yamamoto E, Suzuki H, et al. Fusobacterium in colonic flora and molecular features of colorectal carcinoma[J]. Cancer Res, 2014, 74(5): 1311-1318.
[8] Yamaoka Y, Suehiro Y, Hashimoto S, et al. Fusobacterium nucleatum as a prognostic marker of colorectal cancer in a Japanese population[J]. J Gastroenterol, 2018, 53(4): 517-524.
[9] Mima K, Nishihara R, Qian ZR, et al. Fusobacterium nucleatum in colorectal carcinoma tissue and patient prognosis[J]. Gut, 2016, 65(12): 1973-1980.
[10] Signat B, Roques C, Poulet P, et al. Fusobacterium nucleatum in periodontal health and disease[J]. Curr Issues Mol Biol, 2011, 13(2): 25-36.
[11] Chen YY, Chen Y, Zhang JX, et al. Fusobacterium nucleatum promotes metastasis in colorectal cancer by activating autophagy signaling via the upregulation of CARD3 expression[J]. Theranostics, 2020, 10(1): 323-339.
[12] Yu T, Guo FF, Yu YN, et al. Fusobacterium nucleatum promotes chemoresistance to colorectal cancer by modulating autophagy[J]. Cell, 2017, 170(3): 548-563.
[13] Kostic AD, Gevers D, Pedamallu CS, et al. Genomic analysis identifies association of Fusobacterium with colorectal carcinoma[J]. Genome Res, 2012, 22(2): 292-298.
[14] Xing F, Meng GX, Zhang Q, et al. Ultrasensitive flow sensing of a single cell using graphene-based optical sensors[J]. Nano Lett, 2014, 14(6): 3563-3569.
[15] 张崇磊, 辛自强, 闵长俊, 等. 基于光学表面波的折射率传感成像技术[J]. 光学学报, 2019, 39(1): 0126009. doi:10.3788/AOS201939.0126009. ZHANG Chonglei, XIN Ziqiang, MIN Changjun, et al. Refractive index sensing imaging technology based on optical surface wave[J]. Acta Optica Sinica, 2019, 39(1): 0126009. doi:10.3788/AOS201939.0126009.
[16] 刘雪莲, 冯保华, 林滨, 等. 基于偏振差分干涉技术声表面波检测系统研究[J]. 激光与红外, 2018, 48(10): 1231-1237. LIU Xuelian, FENG Baohua, LIN Bin, et al. Research on SAW detection system based on optical interference technology[J]. Laser & Infrared, 2018, 48(10): 1231-1237.
[17] Chen G, Gao CN, Jiang S, et al. Fusobacterium nucleatum outer membrane vesicles activate autophagy to promote oral cancer metastasis[J]. J Adv Res, 2024, 56: 167-179. doi:10.1016/j.jare.2023.04.002.
[18] Duan CH, Tang XL, Wang WJ, et al. Lactobacillus rhamnosus attenuates intestinal inflammation induced by Fusobacterium nucleatum infection by restoring the autophagic flux[J]. Int J Mol Med, 2021, 47(1): 125-136.
[19] Rubinstein MR, Baik JE, Lagana SM, et al. Fusobacterium nucleatum promotes colorectal cancer by inducing Wnt/β-catenin modulator Annexin A1[J]. EMBO Rep, 2019, 20(4): e47638. doi:10.15252/embr.201847638.
[20] 窦婷婷, 金玲, 池宇欣, 等. 自噬的检测方法及其在相关疾病中的研究进展[J]. 包头医学院学报, 2021, 37(9): 111-116. DOU Tingting, JIN Ling, CHI Yuxin, et al. Detection methods of autophagy and its research progress in related diseases[J]. Journal of Baotou Medical College, 2021, 37(9): 111-116.
[21] Sun LX, Wang YJ, Zhang HQ, et al. Graphene-based confocal refractive index microscopy for label-free differentiation of living epithelial and mesenchymal cells[J]. ACS Sens, 2020, 5(2): 510-518.
[22] Gao YH, Bi DX, Xie RT, et al. Fusobacterium nucleatum enhances the efficacy of PD-L1 blockade in colorectal cancer[J]. Signal Transduct Target Ther, 2021, 6(1): 398. doi:10.1038/S41392-021-00795-x.
[23] Yang YZ, Weng WH, Peng JJ, et al. Fusobacterium nucleatum increases proliferation of colorectal cancer cells and tumor development in mice by activating toll-like receptor 4 signaling to nuclear factor-κB, and up-regulating expression of microRNA-21[J]. Gastroenterology, 2017, 152(4): 851-866.
[24] Chen LF, Zhao R, Shen JJ, et al. Antibacterial Fusobacterium nucleatum-mimicking nanomedicine to selectively eliminate tumor-colonized bacteria and enhance immunotherapy against colorectal cancer[J]. Adv Mater, 2023, 35(45): e2306281. doi:10.1002/adma.202306281.
[25] Hong J, Guo FF, Lu SY, et al. F. nucleatum targets lncRNA ENO1-IT1 to promote glycolysis and oncogenesis in colorectal cancer[J]. Gut, 2021, 70(11): 2123-2137.
[26] Chen SJ, Zhang L, Li MJ, et al. Fusobacterium nucleatum reduces METTL3-mediated m6A modification and contributes to colorectal cancer metastasis[J]. Nat Commun, 2022, 13(1): 1248. doi:10.1038/s41467-022-28913-5.
[27] Zhu HY, Li M, Bi DX, et al. Fusobacterium nucleatum promotes tumor progression in KRAS p.G12D-mutant colorectal cancer by binding to DHX15[J]. Nat Commun, 2024, 15(1): 1688. doi:10.1038/S41467-024-45572-w.

相关文章 15

[1]	王潇,孔文茹,崔伟亮,王姝麒. 羊角棉总生物碱增强氟尿嘧啶对结直肠癌化疗敏感性[J]. 山东大学学报 (医学版), 2024, 62(6): 30-37.
[2]	魏闫若雪,李梓绮,刘春铖,刘晓晗,赵然,刘玉昆. 结直肠癌中SP1的瘤内异质性表达及其临床意义[J]. 山东大学学报 (医学版), 2024, 62(5): 89-94.
[3]	刘爱静,李雁儒,高惠茹,段伟丽,李培龙,李娟,杜鲁涛,王传新. 自噬相关蛋白5在结肠癌中的表达及对结肠癌细胞迁移及侵袭能力的影响[J]. 山东大学学报 (医学版), 2024, 62(4): 14-23.
[4]	刘春铖,刘晓晗,魏闫若雪,李梓绮,刘玉昆,赵然. 结直肠癌中含溴结构域蛋白9的亚细胞定位模式及其临床意义[J]. 山东大学学报 (医学版), 2024, 62(4): 24-30.
[5]	董雅琪,王新慧,赵颖慧,王传新. 血清外泌体LINC02163作为结直肠癌远处转移标志物的临床价值[J]. 山东大学学报 (医学版), 2023, 61(9): 19-28.
[6]	郑荣慧,李攀,曹秀琴,贺瑞霞,陈民佳,陈海霞,杨志伟. SQSTM1蛋白在嗜肺军团菌感染RAW264.7细胞自噬中的作用机制[J]. 山东大学学报 (医学版), 2023, 61(6): 10-21.
[7]	任慧欣,郑茂金,韩文灿,王超群,周云,裴冬生. 过氧化氢通过调控自噬增强宫颈癌的放疗敏感性[J]. 山东大学学报 (医学版), 2023, 61(6): 22-28.
[8]	何静,严如根,武志红,李长忠. 消癥抑癌方对卵巢癌SKOV3细胞增殖、迁移的影响[J]. 山东大学学报 (医学版), 2023, 61(5): 1-10.
[9]	华月帆,何珂瑶,张家豪,钱梦凡,刘怡文,孔金玉,杨海军,周福有. 具核梭杆菌诱导缺氧诱导因子及血管生成因子高表达对食管鳞癌患者生存预后的影响[J]. 山东大学学报 (医学版), 2023, 61(11): 59-67.
[10]	孙富云,王维鹏,张会会,耿艳,安小霞,李双双,张彬彬. 结直肠癌术后患者人格特质与抑郁、焦虑症状的关联性[J]. 山东大学学报 (医学版), 2021, 59(7): 91-96.
[11]	徐兵,李勇,刘明,刘永辉. 沉默PRRX1基因表达可增强前列腺癌耐药细胞株PC-3/DTX对多西他赛的敏感性[J]. 山东大学学报 (医学版), 2021, 59(6): 103-110.
[12]	张华宇,殷思源,刘健,马嘉旭,宋茹,曹国起,王一兵. 氧糖剥夺条件下培养表皮干细胞的定量蛋白质组学分析[J]. 山东大学学报 (医学版), 2021, 59(4): 17-27.
[13]	张小红,周云,杜秋莹,任慧欣,王超群. Atg7-siRNA通过调节精氨酸循环干扰食管癌ECA109细胞放疗敏感性[J]. 山东大学学报 (医学版), 2021, 59(4): 28-34.
[14]	杜甜甜,李娟,赵颖慧,段伟丽,王景,王允山,杜鲁涛,王传新. 长链非编码RNA LINC02474在结直肠癌中的表达特征及对细胞增殖的影响[J]. 山东大学学报 (医学版), 2021, 59(10): 59-69.
[15]	李宁,李娟,谢艳,李培龙,王允山,杜鲁涛,王传新. 长链非编码RNA AL109955.1在80例结直肠癌组织中的表达及对细胞增殖与迁移侵袭的影响[J]. 山东大学学报 (医学版), 2020, 1(7): 38-46.

多维度评价

Viewed

Full text

Abstract

Cited

Shared

Discussed