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中华细胞与干细胞杂志(电子版) ›› 2022, Vol. 12 ›› Issue (02) : 65 -73. doi: 10.3877/cma.j.issn.2095-1221.2022.02.001

论著

过表达DNALI1抑制低氧环境中鼻咽癌细胞株CNE-2z侵袭与迁移
戴炜1, 黄琪琪2, 时悦3, 熊子琪3, 杨甫文4,(), 黄慧芳2,()   
  1. 1. 350001 福州,福建医科大学附属协和医院中心实验室;350004 福州,福建医科大学医学技术与工程学院
    2. 350001 福州,福建医科大学附属协和医院中心实验室
    3. 350004 福州,福建医科大学医学技术与工程学院
    4. 350025 福州,联勤保障部队第九〇〇医院耳鼻咽喉头颈外科
  • 收稿日期:2022-01-29 出版日期:2022-04-01
  • 通信作者: 杨甫文, 黄慧芳
  • 基金资助:
    福建医科大学大学生创新创业训练计划项目(202010392015); 高水平实验研究平台建设项目(闽201704)

DNALI1 inhibits invasion and migration of nasopharyngeal carcinoma CNE-2z cell in a hypoxic environment

Wei Dai1, Qiqi Huang2, Yue Shi3, Ziqi Xiong3, Fuwen Yang4,(), Huifang Huang2,()   

  1. 1. Central Laboratory, Fujian Medical University Union Hospital, Fuzhou 350001, China; College of Medical Technology and Engineering, Fujian Medical University, Fuzhou 350004, China
    2. Central Laboratory, Fujian Medical University Union Hospital, Fuzhou 350001, China
    3. College of Medical Technology and Engineering, Fujian Medical University, Fuzhou 350004, China
    4. Department of Otorhinolaryngology, Head and Neck Surgery, The 900th Hospital of the People's Liberation Army Joint Service Support Force, Fuzhou 350025, China
  • Received:2022-01-29 Published:2022-04-01
  • Corresponding author: Fuwen Yang, Huifang Huang
引用本文:

戴炜, 黄琪琪, 时悦, 熊子琪, 杨甫文, 黄慧芳. 过表达DNALI1抑制低氧环境中鼻咽癌细胞株CNE-2z侵袭与迁移[J]. 中华细胞与干细胞杂志(电子版), 2022, 12(02): 65-73.

Wei Dai, Qiqi Huang, Yue Shi, Ziqi Xiong, Fuwen Yang, Huifang Huang. DNALI1 inhibits invasion and migration of nasopharyngeal carcinoma CNE-2z cell in a hypoxic environment[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2022, 12(02): 65-73.

目的

探讨模拟肿瘤内低氧微环境下,动力蛋白轴突轻链中间体1 (DNALI1)对鼻咽癌细胞株CNE-2z增殖、迁移、侵袭和凋亡的影响。

方法

R语言软件分析GEO数据库中鼻咽癌组织的3个数据集,分析其差异基因(DEGs)。低氧是肿瘤微环境基本特征,体外细胞研究均在低氧工作站(1﹪O2)中进行,以模拟体内低氧微环境。将DNALI1过表达质粒和空载体质粒包装成慢病毒后感染CNE-2z细胞,构建DNALI1基因稳定过表达的CNE-2z细胞株。将CNE-2z细胞分别进行正常培养(空白对照,BC),感染空载体慢病毒(阴性对照,NC)和感染过表达DNALI1慢病毒(过表达DNALI1,DNALI1-OE)。实时荧光定量聚合酶链式反应(RT-qPCR)和Western blot检测DNALI1 mRNA和蛋白表达水平;集落形成实验检测细胞增殖能力;Transwell实验检测细胞迁移和侵袭能力;流式细胞术检测细胞凋亡水平。多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。

结果

生物信息学分析发现,与健康人鼻咽组织相比,鼻咽癌组织中DNALI1基因低表达。在低氧环境中,BC组与NC组细胞增殖、凋亡、迁移和侵袭比较,差异均无统计学意义;与BC组和NC组比较,DNALI1-OE组细胞增殖能力和凋亡水平比较,差异均无统计学意义(P均> 0.05)。与BC组和NC组比较,DNALI1-OE组细胞迁移能力[(198.67±3.22),(199.00±3.00)比(75.00±7.55)个]和侵袭能力[(240.67±16.01),(259.67±3.06)比(83.33±9.50)个]降低,差异具有统计学意义(P均< 0.001)。

结论

低氧环境中,DNALI1过表达可抑制CNE-2z细胞侵袭与迁移能力,但对细胞增殖、凋亡无明显影响。

Objective

To investigate the effects of dynein axonemal light intermediate chain 1 (DNALI1) on proliferation, invasion, migration and apoptosis of CNE-2z cells in a hypoxic environment.

Methods

Three data sets about nasopharyngeal carcinoma tissues in Gene Expression Omnibus (GEO) database were analyzed to find differential expressedgenes (DEGs) through R language software. Hypoxia is the characteristic of tumor microenvironment. This study was carried out in a hypoxia work station (1﹪O2) to simulate the hypoxic microenvironment in vivo. Stable overexpressed DNALI1CNE-2z cells were established by infecting with lentivirus packaged with DNALI1 overexpressed plasmid. CNE-2z cells were cultured normally (blank control, BC) , infected with empty vector virus (negative control, NC) and DNALI1 overexpression vector virus (DNALI1-OE) respectively, and then exposed to hypoxic environment (1﹪O2) . The mRNA and protein expression levels of DNALI1 gene were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blot. The proliferation of CNE-2z cells was measured by colony formation assay. Transwell assay was used to detect the migration and invasion of CNE-2z cells. The apoptotic cells of CNE-2z cells were detected by flow cytometry. One-way ANOVA was used for comparison among multiple groups. LSD-t test was used for pairwise comparison between groups. P values of less than 0.05 were considered statistically significant.

Results

Through bioinformatics analysis, it was found that the expression of DNALI1 was downregulated in nasopharyngeal carcinoma compared with normal nasopharyngeal tissue. After DNALI1 overexpression, the proliferation ability and apoptotic cells of CNE-2z cells remained unchanged in the hypoxic environment compared with BC and NC groups (all P > 0.05) . However, compared with BC group and NC group, the cell migration ability (198.67±3.22) , (199.00±3.00) vs (75.00±7.55) and invasion ability (240.67±16.01) , (259.67±3.06) vs (83.33±9.50) were significantly inhibited in DNALI1-OE group (all P < 0.001) .

Conclusion

DNALI1 overexpression inhibits the invasion and migration of CNE-2z cells in hypoxic environment, but has no effect on proliferation and apoptosis of CNE-2z cells.

表1 引物序列信息
图1 鼻咽癌组织和正常鼻咽组织差异基因的鉴定注:a ~ c图为基因表达情况的火山图;d图为3个数据集的韦恩图
图2 利用R包clusterProfiler对DEGs进行GO和KEGG的富集分析注:a图为生物过程分析;b图为细胞组成分析;c图为分子功能分析;d图为KEGG分析
图3 PPI网络及其重要模块
表2 五种算法筛选前10位的hub基因
图4 DNALI1过表达后检测CNE-2z细胞中DNALI1基因的表达水平注:a图为RT-qPCR检测DNALI1过表达慢病毒和空载体慢病毒感染CNE-2z后各组细胞内DNALI1 mRNA表达水平;b图为Western blot检测DNALI1过表达慢病毒和空载体慢病毒感染CNE-2z后各组细胞内DNALI1蛋白表达水平;与DNALI1-OE组比较,aP < 0.001;ns为差异无统计学意义
图5 DNALI1基因过表达不影响低氧环境中CNE-2z细胞的增殖能力注:a ~ c图为正常视野下集落形成实验分别检测BC组、NC组和DNALI1-OE组CNE-2z细胞的增殖能力;d图为统计分析各组细胞集落形成率的差异;ns为差异无统计学意义
图6 DNALI1基因过表达不影响低氧环境中CNE-2z细胞凋亡水平注:a ~ c图为流式细胞术分别检测BC组、NC组和DNALI1-OE组CNE-2z细胞的凋亡水平;d图为统计分析凋亡细胞百分比差异;ns为差异无统计学意义
图7 DNALI1基因过表达可抑制低氧环境中CNE-2z细胞的迁移能力注:a ~ c图为倒置显微镜下观察BC组、NC组和DNALI1-OE组CNE-2z细胞的迁移能力(×200);d图为统计分析各组细胞穿过transwell小室的细胞数差异;与DNALI1-OE组比较,aP < 0.001;ns为差异无统计学意义
图8 DNALI1基因过表达可抑制低氧环境中CNE-2z细胞的侵袭能力注:a ~ c图为倒置显微镜下观察BC组、NC组和DNALI1-OE组CNE-2z细胞的侵袭能力(×200);d图为统计分析各组细胞穿过transwell小室的细胞数差异;与DNALI1-OE组比较,aP < 0.001;ns为差异无统计学意义
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