具核梭杆菌通过lncRNA XIST促进结直肠癌细胞化疗药物抵抗

doi:10.3969/j.issn.1673-8640.2025.05.003

摘要/Abstract

摘要：

目的探讨具核梭杆菌对人结直肠癌（CRC）细胞化疗药物抵抗的作用。方法选取2015年3月—2017年1月同济大学附属杨浦医院CRC患者87例。收集所有患者的CRC组织并检测具核梭杆菌丰度，选取其中20例根据具核梭杆菌丰度分为Fn组（10例，有具核梭杆菌感染）和NC组（10例，无具核梭杆菌感染），检测15种候选长链非编码RNA（lncRNA）的相对表达量；根据3年随访结果，从87例患者中独立选取复发患者10例（复发组）和未复发患者10例（未复发组），检测目标lncRNA表达差异。根据不同处理方式将CRC细胞系（HCT116和SW480）进行分组。采用CCK-8实验检测各组细胞的活性，计算半数抑制浓度（IC₅₀）值。采用CCK-8实验检测lncRNA XIST敲减前后CRC细胞系增殖活性的变化。采用CCK-8实验、流式细胞术及蛋白免疫印迹法检测lncRNA XIST敲减前后耐药CRC细胞系（HCT116/5-FU、SW480/5-FU）增殖活性和凋亡的变化。结果与5-FU组和OXA组比较，E.coli/5-FU组和E.coli/OXA组IC₅₀值差异均无统计学意义（P>0.05），Fn/5-FU组和Fn/OXA组IC₅₀值均显著升高（P<0.01）。在15个候选lncRNA中，Fn组表达最高的lncRNA为lncRNA XIST，且显著高于NC组（P<0.01）。复发组癌组织中具核梭杆菌丰度和lncRNA XIST相对表达量均显著高于未复发组（P<0.05）。感染具核梭杆菌的CRC细胞系lncRNA XIST相对表达量显著高于对照组（P<0.01）。HCT116/5-FU细胞和SW480/5-FU细胞lncRNA XIST相对表达量显著高于HCT116细胞和SW480细胞（P<0.001）；与对照组比较，lncRNA XIST敲减组细胞活性、IC₅₀值显著降低（P<0.01），凋亡率显著升高（P<0.01），促凋亡蛋白PARP、Caspase-3和Bax表达均显著升高（P<0.01），凋亡抑制蛋白Bcl-2表达显著降低（P<0.01）。结论具核梭杆菌可以通过上调lncRNA XIST表达促进CRC细胞产生化疗药物抵抗，具核梭杆菌和lncRNA XIST或可作为反映CRC化疗药物敏感性的潜在的生物标志物。

关键词: 具核梭杆菌, 长链非编码RNA, 5-氟尿嘧啶, 奥沙利铂, 结直肠癌

Abstract:

Objective To investigate the role of Fusobacterium nucleatum（Fn） in chemoresistance of colorectal cancer（CRC） cells. Methods A total of 87 CRC patients at Yangpu Hospital Affiliated to Tongji University from March 2015 to January 2017 were enrolled. CRC tissues from all the patients were collected to determine Fn abundance. Totally，20 cases were selected and classified into Fn group（10 cases with Fn infection） and NC group（10 cases without Fn infection） based on bacterial load. The relative expression of 15 candidate long non-coding RNA（lncRNA）was determined. Additionally，10 recurrent and 10 non-recurrent patients were independently selected from the 87 patients based on 3-year follow-up outcomes to compare target lncRNA expression levels. CRC cell lines（HCT116 and SW480）were classified according to experimental conditions. Cell viability was assessed by CCK-8 assay to calculate the half-maximal inhibitory concentration（IC₅₀）. Proliferative activity of CRC cell lines before and after lncRNA XIST knockdown was evaluated using CCK-8 assay. CCK-8 assay，flow cytometry and western blotting were used to analyze proliferation and apoptosis in chemoresistant CRC cell lines（HCT116/5-FU and SW480/5-FU）. Results Compared with 5-FU and OXA treatment groups，the E.coli/5-FU and E.coli/OXA groups showed no statistical significance in IC₅₀（P>0.05），whereas Fn/5-FU and Fn/OXA groups exhibited increased IC₅₀（P<0.01）. Among the 15 candidate lncRNA，lncRNA XIST showed the highest expression in Fn group，exceeding NC group（P<0.01）. Both Fn abundance and lncRNA XIST expression levels were higher in recurrence group than non-recurrence group（P<0.05）. Fn-infected CRC cell lines demonstrated increased lncRNA XIST expression compared with control group（P<0.01）. HCT116/5-FU and SW480/5-FU showed higher lncRNA XIST relative expression than HCT116 and SW480 cells（P<0.001）. The lncRNA XIST knockdown group showed reduced cell viability and IC₅₀（P<0.01），increased apoptosis rate（P<0.01），upregulated apoptosis protein（PARP，Caspase-3 and Bax）（P<0.01）and downregulated anti-apoptotic protein Bcl-2（P<0.01）. Conclusions Fn may promote chemoresistance in CRC cells through lncRNA XIST upregulation. Both Fn and lncRNA XIST could serve as potential biomarkers for predicting chemotherapy sensitivity in CRC patients.

Key words: Fusobacterium nucleatum, Long non-coding RNA, 5-Fluorouracil, Oxaliplatin, Colorectal cancer

中图分类号:

R446.1

吴亚洲, 李汉华, 孟磊俊, 陈学飞, 孙钰涵, 翁文浩, 郑冰. 具核梭杆菌通过lncRNA XIST促进结直肠癌细胞化疗药物抵抗[J]. 检验医学, 2025, 40(5): 428-436.

WU Yazhou, LI Hanhua, MENG Leijun, CHEN Xuefei, SUN Yuhan, WENG Wenhao, ZHENG Bing. Fusobacterium nucleatum promoting chemoresistance of colorectal cancer cells via lncRNA XIST[J]. Laboratory Medicine, 2025, 40(5): 428-436.

图/表 12

参考文献 32

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引物名称	正向引物（5'~3'）	反向引物（5'~3'）
siNC	UUCUCCGAACGUGUCACGUTT	ACGUGACACGUUCGGAGAATT
siXIST	GCUGACUACCUGAGAUUUATT	UAAAUCUCAGGUAGUCAGCTT

引物名称	正向引物（5'~3'）	反向引物（5'~3'）
siNC	UUCUCCGAACGUGUCACGUTT	ACGUGACACGUUCGGAGAATT
siXIST	GCUGACUACCUGAGAUUUATT	UAAAUCUCAGGUAGUCAGCTT

引物名称	正向引物（5'~3'）	反向引物（5'~3'）
具核梭杆菌	GCTTGAAATGGAAGCTACAAGAGA	GGGTCAGAACCAACTCCTACAA
PGT	ATCCCCAAAGCACCTGGTTT	AGAGGCCAAGATAGTCCTGGTAA

引物名称	正向引物（5'~3'）	反向引物（5'~3'）
具核梭杆菌	GCTTGAAATGGAAGCTACAAGAGA	GGGTCAGAACCAACTCCTACAA
PGT	ATCCCCAAAGCACCTGGTTT	AGAGGCCAAGATAGTCCTGGTAA

引物名称	正向引物（5'~3'）	反向引物（5'~3'）
β-actin	CATGTACGTTGCTATCCAGGC	CTCCTTAATGTCACGCACGAT
lncRNA XIST	GGAAAAGTGGCCGCCATTTT	ACATATGACAACGCCTGCCA
lncRNA snaR	CACGTGGTGGCAAAGGAAAG	TCGTGACTTCCATTGTGGGG
lncRNA UCA1	TCCTTCCAAATGGCCATCCC	ACCAAAGCCTTTTGTCCCCA
lncRNA SLC25A25-AS1	GCTTGGACTTCCTGGAGGAC	GGAGGAGGCAGGAGAGAGAA
Linc00152	TCTCCTTGGCTTGCAGATGG	GACTGGCCAGACAAATGGGA
lncRNA H19	AGACACCATCGGAACAGCAG	ATGATGAGTCCAGGGCTCCT
lncRNA MALAT1	GGACTTGCCTCAACTCCCTC	GCCCTCTCAGCCACTCAAAT
lncRNA CRNDE	GGCGCTAACGGTCGGTAAC	TTCTGCGTGACAACTGAGGA
lncRNA MEG3	GGCCTGTCTACACTTGCTGT	CAACAGCCCTGTGAGGTAGG
lncRNA PCAT-1	AAACCGCAACATCACCAACG	TTCACCGTGTTAGCCAGGAC
Linc00261	AGGATTCTGCATGTGGGTGG	GCCTTTGGGGAAATGCTTGG
lncRNA PVT1	TGAGGAGCTGCATCTACCCT	GGGTGGACAGGTAACAGGTG
lncRNA MIR100HG	GGGCTGCGGAATTTTGGAAG	TTGCACTGGGGAGACATTCC
lncRNA TUG1	TGGTATGGCTGGCCTTTCTG	CTGAGAGCCCAACTGTCCTG
lncRNA HOTAIR	GGAAGCGAAGGGGTTGTGTA	GCAGGAGGAAGTTCAGGCAT