国际口腔医学杂志

国际口腔医学杂志 ›› 2025, Vol. 52 ›› Issue (4): 473-483.doi: 10.7518/gjkq.2025062

• 论著 • 上一篇    下一篇

微小RNA200a/141-信号转导和转录激活因子4轴在口腔鳞状细胞癌进展中的作用研究

姚曼曼(),仇永乐(),刘铁军,路月亭,路华林,尚宏悦,董博   

  1. 河北医科大学第四医院口腔科 石家庄 050000
  • 收稿日期:2024-06-10 修回日期:2025-01-08 出版日期:2025-07-01 发布日期:2025-06-20
  • 通讯作者: 仇永乐
  • 作者简介:姚曼曼，主治医师，硕士，Email：49103567@hebmu.edu.cn
  • 基金资助:
    河北省中医药管理局项目(2024050);河北省自然科学基金面上项目(H2024206476);中央引导地方科技发展资金项目(246Z7762G)

Role of mircoRNA200a/141-signal transduction and transcriptional activator 4 axis in the progression of oral squamous cell carcinoma

Manman Yao(),Yongle Qiu(),Tiejun Liu,Yueting Lu,Hualin Lu,Hongyue Shang,Bo Dong   

  1. Dept. of Stomatology, the Fourth Hospital, Hebei Medical University, Shijiazhuang 050000, China
  • Received:2024-06-10 Revised:2025-01-08 Online:2025-07-01 Published:2025-06-20
  • Contact: Yongle Qiu
  • Supported by:
    Project of the Hebei Provincial Administration of Traditional Chinese Medicine(2024050);Hebei Provincial Natural Science Foundation General Project(H2024206476);Central Government Guides Local Funds Supported by S&T Program of Hebei(246Z7762G)

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摘要：

目的 基于公共数据库探索微小RNA（miRNA）200a/141、信号转导和转录激活因子4（STAT4）在口腔鳞状细胞癌（OSCC）中的表达及临床意义并构建预后模型，探讨其作为潜在治疗靶点的可行性。通过体外实验验证仿生明胶外泌体颗粒递送miRNA200a/141进而调节STAT4，评估在OSCC的细胞和分子水平上的核酸保护能力及抗癌作用。 方法 通过癌症基因组图谱数据库下载OSCC相关miRNA及信使RNA表达测序数据，使用R语言进行数据分析，评估miRNA200a/141与STAT4的表达水平和临床相关性。制备负载miRNA200a/141的仿生明胶外泌体纳米颗粒（GNP-EXO-miRNA200a/141），进行Transwell迁移、细胞划痕和四甲基偶氮唑蓝实验，评估纳米颗粒对SCC25细胞的作用，并通过定量聚合酶链式反应和蛋白质免疫印迹法检测其对SCC25细胞中STAT4表达的影响。 结果 miRNA200a/141在OSCC组织中的表达水平显著降低，而其靶标STAT4的表达水平升高，两者呈负相关。制备的GNP-EXO-miRNA200a/141纳米颗粒具有良好的粒径分布和稳定性。在细胞实验中，GNP-EXO-miRNA200a/141显著抑制了SCC25细胞的增殖和迁移，并显著下调了STAT4的表达。 结论 miRNA200a/141通过调节STAT4在OSCC中发挥重要作用，其有望成为OSCC的诊断、治疗和预后的潜在的分子标志物和治疗靶点。

关键词: 口腔鳞状细胞癌, 微小RNA 200a/141, 信号转导和转录激活因子4, 外泌体, 明胶纳米颗粒

Abstract:

Objective　This study aimed to explore the expression and clinical significance of mircoRNA (miRNA) 200a/141 and signal transducer and activator of transcription 4 (STAT4) in oral squamous cell carcinoma (OSCC) and construct a prognostic model to explore its feasibility as a potential therapeutic target. In vitro experiments verified that bionic gelatin exosome particles delivered mi-RNA200a/141 to regulate STAT4. This study also aimed to evaluate the protective capacity and anticancer effects of nucleic acids at the cellular and molecular levels of OSCC. Methods　OSCC-related miRNA-sequencing and messenger RNA-sequencing data were downloaded from The Cancer Genome Atlas database. Data analysis was performed using R language to assess the expression levels and clinical correlation of miRNA200a/141 with STAT4. Biomimetic gelatin exosome nanoparticles (GNP-EXO-mi-RNA200a/141) loaded with miRNA were prepared, and Transwell migration assay, cell scratch assay, and methyl thiazolyl tetrazolium assay were performed to evaluate the effect of nanoparticles on SCC25 cells. The effects of the nanoparticles on STAT4 expression in SCC25 cells were examined using quantitative polymerase chain reaction and Western blot. Results　The expression of miRNA200a/141 was significantly reduced in OSCC tissues, whereas that of its target STAT 4 increased, which was inversely correlated. The prepared GNP-EXO-miRNA200a/141 nanoparticles showed good size distribution and stability. In cell experiments, GNP-EXO-miRNA200a/141 significantly inhibited the proliferation and migration of SCC25 cells and significantly downregulated STAT4 expression. Conclusion　miRNA200a/141 plays an important role in OSCC by regulating STAT4, and it is expected to be a potential molecular marker and therapeutic target for the diagnosis, treatment, and prognosis of OSCC.

Key words: oral squamous cell carcinoma, mircoRNA200a/141, signal transducer and activator of transcription 4, exosomes, gelatin nanoparticles

中图分类号: 

  • R782

图 1

仿生明胶EXO颗粒的制备模式图"

表 1

引物序列"

基因引物
STAT4

正向引物，5’-AGCCATCTCGGAGGAATA-3’

反向引物，5’-CAGACAACCGGCCTTTAT-3’

GAPDH

正向引物，5’-CGTGGGCCGCCCTAGGCACCA-3’

反向引物，5’-TTGGCTTAGGGTTCAGGGGGG-3’

miRNA-200a

正向引物，5’-TAACACTGTCTGGTAACGATGT-3’

反向引物，5’-ATCGTTACCAGACAGTGTTATT-3’

miRNA-141

正向引物，5’-ACACTCCAGCTGGTAACACTGTCTGGTAA-3’

反向引物，5’-CTCAACTGGTGTCGTGGAGTCGGCA-ATTCAGTT GAGCCATCTTT-3’

图 2

单基因差异分析A：OSCC组织中miRNA-200a表达水平；B：OSCC组织中miRNA-141表达水平；C：OSCC组织中STAT4表达水平。"

图 3

miRNA-200a/141和STAT4相关性A：miRNA-200a；B：miRNA-141。"

图 4

miRNA-200a/141和STAT4表达与OSCC患者预后的生存分析A：miRNA-200a（P=0.006 5）；B：miRNA-141（P=0.001）；C：STAT4（P<0.000 1）。"

表 2

影响OSCC预后的单因素多因素分析"

特征例数单因素分析多因素分析
风险比（95%置信区间）P值风险比（95%置信区间）P值
年龄/岁<5044参照
≥502151.186（0.691~2.038）0.536
性别男176参照
女831.209（0.805~1.815）0.360
分期Ⅰ期和Ⅱ期64参照
Ⅲ期和Ⅳ期1951.776（1.086~2.905）0.022
T分期T1~T2111参照参照
T3~T41482.269（1.464~3.517）<0.0011.863（1.201~2.891）0.006
N分期N0104参照参照
N1460.526（0.243~1.137）0.1020.574（0.265~1.241）0.158
N2~N31091.847（1.206~2.829）0.0051.451（0.943~2.232）0.090
miRNA-200a高表达82参照
低表达1771.916（1.193~3.076）0.007
miRNA-141高表达86参照参照
低表达1732.298（1.377~3.835）0.0012.077（1.240~3.478）0.005
STAT4低表达153参照参照
高表达1063.016 （2.017~4.511）<0.0012.715（1.809~4.075）<0.001

图 5

GNP-EXO和GNP-EXO-miRNA200a/141特征图A：GNP-EXO的TEM图像 × 40 000；B：GNP-EXO-miRNA200a/141的TEM图像 × 40 000；C：GNP-EXO的尺寸分布；D：GNP-EXO-miRNA200a/141的尺寸分布；E：MSC、GNP-EXO、GNP-EXO-miRNA200a/141样本中CD63和CD9的表达；F：GNP-EXO-miRNA200a/141对miRNA的保护作用。"

表 3

纳米粒子的表征"

组别粒径/nmPDI表面电位/mV
GNP109.1±12.10.193±0.05024.53±0.70
GNP-EXO123.1±12.30.216±0.080-11.00±1.20
GNP-EXO-miRNA200a/141127.3±14.00.214±0.030-10.20±0.70

图6

GNP-EXO-miRNA200a/141颗粒对SCC25细胞生长的抑制作用24、48、72 h的GNP-EXO、miRNA200a/141和GNP-EXO-mi-RNA 200a/141处理SCC25后的细胞活力；*P<0.05，**P<0.01。"

图 7

Transwell实验结果A：Transwell实验检测细胞迁移能力，与对照组相比，*P<0.05，**P<0.01，1是GNP-EXO，2是miRNA200a/141，3是GNP-EXO-miRNA200a/141；B：GNP-EXO 结晶紫染色 × 100；C：miRNA200a/141 结晶紫染色 × 100；D：GNP-EXO-miRNA200a/141 结晶紫染色 × 100。"

图8

细胞划痕实验结果"

图 9

SCC25细胞中STAT4蛋白的表达A：qRT-PCR实验；B：蛋白质印迹实验柱状图；C：蛋白质印迹实验电泳图；与对照组相比，*P<0.05，**P<0.01；1是GNP-EXO，2是mi-RNA200a/141，3是GNP-EXO-miRNA200a/141。"

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