国际肿瘤学杂志

国际肿瘤学杂志 ›› 2018, Vol. 45 ›› Issue (11): 641-646.doi: 10.3760/cma.j.issn.1673-422X.2018.11.001

• 论著 •    下一篇

miR-205-3p、miR-205-5p对头颈鳞状细胞癌细胞增殖、迁移及侵袭的影响

余长云，刘勇，曹华   

  1. 450052 郑州大学第一附属医院耳鼻咽喉科（余长云、曹华）；中南大学湘雅医院耳鼻咽喉头颈外科（刘勇）
  • 出版日期:2018-11-08 发布日期:2018-12-21
  • 通讯作者: 曹华，Email: chcaren@163.com E-mail:chcaren@163.com
  • 基金资助:
    国家自然科学基金（81402232）

Effects of microRNA-205-3p and microRNA-205-5p on the cell proliferation, migration and invasion of head and neck squamous cell carcinoma

Yu Changyun, Liu Yong, Cao Hua   

  1. Department of Otolaryngology, First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China
  • Online:2018-11-08 Published:2018-12-21
  • Contact: Cao Hua, Email: chcaren@163.com E-mail:chcaren@163.com
  • Supported by:
    National Natural Science Foundation of China (81402232)

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摘要： 目的 观察微小RNA-205（miR-205）-3p及miR-205-5p对头颈部鳞状细胞癌细胞增殖、迁移及侵袭的影响。方法 下载癌症基因组图集中头颈鳞状细胞癌miRNA表达数据，分析miR-205在头颈鳞状细胞癌组织及癌旁组织中的表达。实时荧光定量聚合酶链反应（qRT-PCR）检测miR-205-3p、miR-205-5p在头颈鳞状细胞癌细胞株5-8F、6-10B、CNE2、Tu686和鼻咽部黏膜组织中的表达水平；miR-205-3p、miR-205-5p抑制物及阴性对照分别转染头颈鳞状细胞癌细胞Tu686，qRT-PCR检测miR-205-3p、miR-205-5p沉默效率，后续实验分为miR-205-3p沉默组及阴性对照组、miR-205-5p沉默组及阴性对照组；CCK-8、Transwell迁移及侵袭实验检测4组细胞的增殖、迁移及侵袭能力。结果 miR-205在头颈鳞状细胞癌组织中的表达显著高于癌旁组织，其中位数（四分位数间距）分别为61 012（51 448）、28 579（35 959），差异有统计学意义（Z=-6.420，P<0.001）。头颈鳞状细胞癌细胞株5-8F、6-10B、CNE2、Tu686和鼻咽部黏膜组织中miR-205-3p表达量分别为0.36±0.07、0.20±0.06、0.15±0.04、0.25±0.04和1.00±0.00，差异有统计学意义（F=162.71，P<0.001）。5-8F、6-10B、CNE2、Tu686和鼻咽部黏膜组织中miR-205-5p表达量分别为0.20±0.01、0.21±0.01、1.06±0.18、23.61±2.07和1.00±0.00，差异有统计学意义（F=371.81，P<0.001）。与5-8F、6-10B、CNE2细胞相比，Tu686细胞中miR-205-3p的表达量高于6-10B、CNE2细胞（P=0.195；P=0.020），低于5-8F细胞（P=0.023）；Tu686细胞中miR-205-5p的表达量最高（P<0.001；P<0.001；P<0.001）。转染抑制物后Tu686细胞中miR-205-3p、miR-205-5p表达量均显著下调，沉默效率分别为87%、83%。沉默miR-205-3p组细胞在培养第1、2、3、4、5天的A值分别为0.26±0.06、0.55±0.11、1.52±0.13、1.91±0.07、2.14±0.24，对照组分别为0.29±0.07、0.78±0.11、1.59±0.15、1.95±0.08、2.02±0.12，两组比较差异均无统计学意义（t=0.506，P=0.639；t=2.459，P=0.070；t=0.573，P=0.597；t=0.655，P=0.548；t=-0.759，P=0.490）；沉默miR-205-5p组细胞在培养第1、2、3、4、5天的A值分别为0.30±0.08、0.61±0.08、0.85±0.08、1.08±0.12、1.16±0.18，对照组分别为0.41±0.10、0.78±0.14、1.33±0.28、1.87±0.09、2.08±0.19，沉默组细胞在培养第3、4、5天的增殖能力均明显低于对照组，差异均有统计学意义（t=3.665，P=0.017；t=12.223，P<0.001；t=7.825，P<0.001）。迁移实验表明，miR-205-3p表达下调后，头颈鳞状细胞癌细胞Tu686的迁移能力无明显变化，沉默组与对照组细胞穿膜数分别为（192.00±28.49）、（188.40±22.52）个，两组比较差异无统计学意义（t=-0.160，P=0.877）；miR-205-5p表达下调后，头颈鳞状细胞癌细胞Tu686的迁移能力明显减弱，沉默组与对照组细胞穿膜数分别为（109.40±27.63）、（183.60±31.63）个，两组比较差异有统计学意义（t=3.951，P=0.004）。侵袭实验表明，miR-205-3p沉默组细胞穿膜数为（93.40±10.24）个，对照组细胞穿膜数为（96.20±16.56）个，两组比较差异无统计学意义（t=0.322，P=0.756）；miR-205-5p沉默组细胞穿膜数为（53.00±17.80）个，对照组细胞穿膜数为（94.40±14.38）个，两组差异有统计学意义（t=4.045，P=0.004）。结论 沉默miR-205-5p可抑制头颈鳞状细胞癌细胞Tu686的增殖、迁移及侵袭能力；沉默miR-205-3p对Tu686细胞增殖、迁移及侵袭能力无明显影响。

关键词: 头颈部肿瘤, 微RNAs, 细胞增殖, 细胞运动, 肿瘤侵润

Abstract: Objective To investigate the effects of microRNA-205-3p (miR-205-3p) and microRNA-205-5p (miR-205-5p) on the cell proliferation, migration and invasion of head and neck squamous cell carcinoma (HNSCC). Methods The miRNA expression data were obtained from The Cancer Genome Atlas, and were used to determine miR-205 expression in HNSCC and paracancerous tissues. Quantitative real-time fluorescent polymerase chain reaction (qRT-PCR) was used to detect the expressions of miR-205-3p and miR-205-5p in HNSCC cell lines 5-8F, 6-10B, CNE2, Tu686 and mucosal tissues of nasopharynx. MiR-205-3p inhibitor, miR-205-5p inhibitor and miR-NC were transfected into Tu686 cells, and qRT-PCR was employed to evaluate the silence efficiency. In the following study, the cells were divided into four groups: miR-205-3p inhibitor and control, miR-205-5p inhibitor and control. Then the cell counting kit (CCK-8) assay, Transwell migration and invasion assays were carried out to examine the proliferation, migration and invasion abilities of the cells in the four groups. Results Compared with paracancerous tissues, the higher expression of miR-205 in HNSCC tissues was observed ［M (QR): 61 012 (51 448) vs. 28 579 (35 959), Z=-6.420, P<0.001)］. The expression levels of miR-205-3p in HNSCC cell lines 5-8F, 6-10B, CNE2, Tu686 and mucosal tissues of nasopharynx were 0.36±0.07, 0.20±0.06, 0.15±0.04, 0.25±0.04 and 1.00±0.00 respectively, with a significant difference (F=162.71, P<0.001). The expression levels of miR-205-5p in cell lines 5-8F, 6-10B, CNE2, Tu686 and mucosal tissues of nasopharynx were 0.20±0.01, 0.21±0.01, 1.06±0.18, 23.61±2.07 and 1.00±0.00 respectively, with a significant difference (F=371.81, P<0.001). Compared with 5-8F, 6-10B, CNE2 cells, the expression of miR-205-3p in Tu686 cells was higher than those in 6-10B, CNE2 cells (P=0.195; P=0.020), and lower than that in 5-8F cells (P=0.023), and the expression of miR-205-5p in Tu686 cells was the highest (P<0.001; P<0.001; P<0.001). The expressions of miR-205-3p and miR-205-5p in Tu686 cells were effectively downregulated by inhibitors, and the silence efficiencies were 87% and 83%, respectively. The absorbance (A) values of miR-205-3p inhibitor group on the first, second, third, fourth and fifth day were 0.26±0.06, 0.55±0.11, 1.52±0.13, 1.91±0.07, 2.14±0.24, and those of miR-NC group were 0.29±0.07, 0.78±0.11, 1.59±0.15, 1.95±0.08, 2.02±0.12. There were no significant differences between the two groups (t=0.506, P=0.639; t=2.459, P=0.070; t=0.573, P=0.597; t=0.655, P=0.548; t=-0.759, P=0.490). The cell proliferations of miR-205-5p inhibitor group on the first, second, third, fourth and fifth day were 0.30±0.08, 0.61±0.08, 0.85±0.08, 1.08±0.12, 1.16±0.18, and those of miR-NC group were 0.41±0.10, 0.78±0.14, 1.33±0.28, 1.87±0.09, 2.08±0.19. The cell proliferations of miR-205-5p inhibitor group on the third, fourth and fifth day were significantly lower than those of miR-NC group (t=3.665, P=0.017; t=12.223, P<0.001; t=7.825, P<0.001). Transwell migration assay demonstrated that downregulation of miR-205-3p had no significant effect on migration abilities of Tu686 cells. The numbers of cell permeating septum of miR-205-3p inhibitor group and miR-NC group were 192.00±28.49 and 188.40±22.52, respectively. There was no significant difference between the two groups (t=-0.160, P=0.877). Downregulation of miR-205-5p significantly decreased the migration abilities of Tu686 cells. The numbers of cell permeating septum of miR-205-5p inhibitor group and miR-NC group were 109.40±27.63 and 183.60±31.63, respectively, and the difference was statistically significant (t=3.951, P=0.004). Transwell invasion assay showed that the numbers of cell permeating septum of miR-205-3p inhibitor group and miR-NC group were 93.40±10.24 and 96.20±16.56, respectively. There was no significant difference between the two groups (t=0.322, P=0.756). The numbers of cell permeating septum of miR-205-5p inhibitor group and miR-NC group were 53.00±17.80 and 94.40±14.38, respectively, and the difference was statistically significant (t=4.045, P=0.004). Conclusion Downregulation of miR-205-5p can inhibit the proliferation, migration and invasion abilities of Tu686 cells. However, downregulation of miR-205-3p has no significant effect on the proliferation, migration and invasion of Tu686 cells.

Key words: Head and neck neoplasms, MicroRNAs, Cell proliferation, Cell movement, Neoplasm invasiveness