天津医药

天津医药 ›› 2021, Vol. 49 ›› Issue (2): 113-118.doi: 10.11958/20200999

• 细胞与分子生物学 •    下一篇

尼克酰胺-N-甲基转移酶在肺腺癌间质中的表达及其生物学功能 #br#

鞠薇薇,于生金,林黎娟,邵志翔,张奇芳,江黎黎   

  1. 1辽东学院医学院分子医学研究室（邮编118003）；2丹东市第一医院病理科
  • 收稿日期:2020-04-14 修回日期:2020-10-22 出版日期:2021-02-15 发布日期:2021-02-02
  • 通讯作者: 张奇芳 E-mail:458570579@qq.com
  • 基金资助:
    辽宁省教育厅自然科学研究基金;辽宁省博士科研启动基金

Expression and biological function of NNMT in stroma of lung adenocarcinoma #br#

JU Wei-wei, YU Sheng-jin, LIN Li-juan, SHAO Zhi-xiang, ZHANG Qi-fang, JIANG Li-li   

  1. 1 Laboratory of Molecular Medicine, School of Medicine, Liaodong University, Dandong 118003, China;
    2 Department of Pathology, Dandong First Hospital
  • Received:2020-04-14 Revised:2020-10-22 Published:2021-02-15 Online:2021-02-02

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摘要： 目的 探讨尼克酰胺-N-甲基转移酶（NNMT）在肺腺癌间质中的表达水平及过表达NNMT对肺腺癌细胞 生物学功能的影响。方法 （1）收集150例肺腺癌患者手术切除的组织标本。免疫组织化学染色检测癌组织和间质 细胞中 NNMT 的表达水平，并分析其表达与患者临床病理特征及预后间的关系。（2）分别转染过表达 NNMT 质粒 （NNMT组）和对照质粒（control组）构建稳定表达NNMT的肺成纤维细胞系HFL-1，采用荧光定量PCR（qPCR）、细胞 免疫荧光、蛋白质印迹法（Western blot）检测NNMT表达水平。（3）收集过表达NNMT及control组HFL-1细胞的培养 上清，分别和肺腺癌A549、PC9细胞进行共培养，通过细胞增殖实验、平板克隆形成实验、Transwell侵袭实验以及划 痕愈合实验比较2种培养液对A549、PC9细胞增殖、克隆形成、迁移及侵袭能力的影响。结果 （1）免疫组化染色显 示，150例患者组织标本中65例癌细胞中NNMT呈高表达，83例间质细胞中NNMT呈高表达。有淋巴结转移和病理 分期Ⅲ~Ⅳ期患者肿瘤间质细胞中NNMT高表达比例较高，且肿瘤间质中高表达NNMT患者的总生存率明显低于低 表达者。（2）成功构建了稳定过表达 NNMT 的 HFL-1 细胞，Western blot、qPCR、细胞免疫荧光染色均提示 NNMT 组 NNMT表达高于control组。（3）细胞共培养结果显示，与control组相比，表达NNMT的肺成纤维细胞系的培养液可以 显著促进A549和PC9细胞的增殖、克隆形成、迁移以及侵袭。结论 肿瘤间质表达的NNMT与肺腺癌的恶性进展密 切相关，可能是肿瘤靶向生物治疗的潜在靶点。

关键词: 肺肿瘤, 腺癌, 烟酰胺N-甲基转移酶, 预后, 间质细胞, A549细胞, 细胞系, 肿瘤, 肿瘤浸润, 细胞运动

Abstract:

Objective To investigate the expression level of nicotinamide N-methyltransferase (NNMT) in the stroma
of lung cancer, and the influence of overexpressed NNMT on the biological function of lung adenocarcinoma cells.
 Methods(1) Tissue specimens were collected from 150 patients with lung adenocarcinoma. Immunohistochemical staining
was used to detect the NNMT expression level in cancer tissues and stromal cells, and the relationship between NNMT
expression and the clinicopathological characteristics and prognosis of the patients were analyzed. (2) A lung fibroblast cell
line HFL-1 with stable NNMT expression was constructed by the transfection of overexpressed NNMT plasmid (NNMT
group) and control plasmid (control group) respectively. The expression levels of NNMT were detected by real-time
fluorescence quantitative PCR (qPCR), immunofluorescence and Western blot assay. (3) The culture supernatant of the overexpressed NNMT and the control group of HFL-1 cells were collected and co-cultured with lung adenocarcinoma A549 and
PC9 cells, respectively. Then, the effects of the two kind of culture supernatant on the proliferation, clonal formation and
migration of A549 and PC9 cells were compared through cell proliferation experiment, plate clonal formation experiment,
scratch healing experiment and Transwell migration experiment. Results(1) Immunohistochemical staining showed that
NNMT was highly expressed in 65 cancer cell samples and 83 mesenchymal cell samples of 150 patients. The NNMT
expression was higher in patients with lymph node metastasis and pathological stage Ⅲ-Ⅳ. The overall survival rate was significantly lower in patients with high expression of NNMT in tumor stroma than those with low expression of NNMT. (2)
HFL-1 cells with stable overexpression of NNMT were successfully constructed. Western blot assay, qPCR and cell
immunofluorescence staining indicated that the NNMT expression was higher in the NNMT group than that in the control
group. (3) Cell co-culture results showed that compared with the control group, the culture medium of lung fibroblast cell
line expressing NNMT could significantly promote the proliferation, clone formation and migration of A549 and PC9 cells.
 ConclusionNNMT is closely related to the malignant progression of lung adenocarcinoma, which may be a potential target
for tumor-targeted biological therapy.

Key words: lung neoplasms, adenocarcinoma, nicotinamide N-methyltransferase, prognosis, stromal cells, A549 cells,
cell line, tumor, neoplasm invasiveness, cell movement

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