国际肿瘤学杂志

国际肿瘤学杂志 ›› 2019, Vol. 46 ›› Issue (2): 77-81.doi: 10.3760/cma.j.issn.1673-422X.2019.02.003

• 论著 • 上一篇    下一篇

miR-193b表达上调对HBV阳性肝癌细胞的影响

 黄锐1, 伍刚1, 许健1, 郑波1, 黄凌远2, 钟振东1   

  1. 1四川省医学科学院 四川省人民医院肝胆外科，成都610072；2成都里来生物科技有限公司分子生物检测部610000
  • 出版日期:2019-02-08 发布日期:2019-04-03
  • 通讯作者: 钟振东，Email: 1505214952@qq.com E-mail:1505214952@qq.com
  • 基金资助:

    四川省卫生和计划生育委员会课题（17PJ587）

Effect of up-regulation of miR-193b on HBV positive hepatocellular carcinoma cells

Huang Rui1, Wu Gang1, Xu Jian1, Zheng Bo1, Huang Lingyuan2, Zhong Zhendong1   

  1. 1Department of Hepatobiliary Surgery, Sichuan Provincial People′s Hospital, Sichuan Academy of Medical Sciences, Chengdu 610072, China;2Department of Molecular Biology, Chengdu Lilai Biotechnology Limited Company, Chengdu 610000, China
  • Online:2019-02-08 Published:2019-04-03
  • Contact: Zhong Zhendong, Email: 1505214952@qq.com E-mail:1505214952@qq.com
  • Supported by:

    Found of Health and Family Planning Commission of Sichuan Province of China (17PJ587)

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摘要： 目的  探讨微小RNA-193b（miR-193b）表达上调对乙型肝炎病毒（HBV）阳性肝癌细胞凋亡、侵袭和转移的影响。方法  HepG2.2.15细胞株培养至对数生长期后，以25、50、100 pmol的miR193b mimic进行转染，对照组以空白mimic进行转染（0 pmol）。48 h后采用实时定量荧光聚合酶链反应（qRT-PCR）检测各组细胞中miR-193b的表达，流式细胞仪检测各实验组细胞凋亡率，细胞划痕实验检测细胞迁移能力，Transwell实验检测细胞侵袭能力，Western blotting检测Bax、Bcl-2、基质金属蛋白酶-9（MMP-9）和基质金属蛋白酶-2（MMP-2）蛋白表达。结果  qRT-PCR结果显示，miR-193b mimic（25、50、100 pmol）转染组和对照组HepG2.2.15细胞株中miR-193b表达量分别为1.05±0.09、1.53±0.12、2.08±0.17和0.49±0.12，4组间差异有统计学意义（F=261.35，P<0.001），各转染组与对照组相比，miR-193b表达明显增加（P=0.036；P=0.029；P=0.022）。流式细胞仪检测结果显示，miR-193b mimic（25、50、100 pmol）转染组和对照组HepG2.2.15细胞凋亡率分别为（30.28±5.22）%、（53.41±6.18）%、（79.89±7.13）%和（1.02±0.13）%，4组间差异有统计学意义（F=357.19，P<0.001），各转染组与对照组相比，细胞凋亡率明显增强（P=0.025；P=0.010；P=0.007）。细胞划痕实验结果显示，miR-193b mimic（25、50、100 pmol）转染组和对照组细胞迁移距离分别为（27.53±1.54）mm、（19.24±2.12）mm、（13.42±1.53）mm和（34.95±1.92）mm，4组间差异有统计学意义（F=408.62,P<0.001），各转染组与对照组相比，细胞迁移距离明显减小（P=0.032；P=0.007；P=0.006）。Transwell实验结果显示，miR193b mimic（25、50、100 pmol）转染组和对照组细胞吸光度值分别为1.02±0.12、0.59±0.13、0.42±0.10和1.68±0.16，4组间差异有统计学意义（F=511.68,P<0.001），各转染组与对照组相比，细胞侵袭能力明显减弱（P=0.028；P=0.005；P=0.002）。Western blotting检测结果显示，miR193b mimic（25、50、100 pmol）转染组和对照组HepG2.2.15细胞Bax、Bcl-2、MMP-9和MMP-2蛋白表达差异均有统计学意义（F=264.38，P<0.001；F=437.19，P<0.001；F=377.46，P<0.001；F=208.79，P<0.001），进一步两两比较，各转染组与对照组Bax、Bcl-2、MMP9和MMP2蛋白表达差异均有统计学意义（均P<0.05）。结论  miR-193b可诱导HBV阳性肝癌细胞凋亡，抑制HBV阳性肝癌细胞侵袭和转移，其机制可能与调控Bax、Bcl-2、MMP-9、MMP-2等蛋白表达有关。

关键词: 肝炎病毒, 乙型, 肝肿瘤, 微RNAs, 肿瘤浸润

Abstract: Objective  To investigate the effect of miRNA-193b (miR-193b) up-regulation on the apoptosis, invasion and metastasis of hepatitis B virus (HBV)positive hepatocellular cells. Methods  HepG2.2.15 cells were cultured until logarithmic growth phase and transfected with miR-193b mimic at a concentration of 25, 50, 100 pmol. The control group was transfected with blank mimic (0 pmol). After 48 hours, the expression of miR-193b in each group was detected by realtime quantitative polymerase chain reaction (qRT-PCR), the apoptosis rate in each group was detected by flow cytometry, the cell migration was detected by cell scratch assay, the cell invasion was detected by Transwell assay, and the expressions of Bax, Bcl-2, matrix metalloprotease (MMP)-9 and MMP-2 protein were detected by Western blotting. Results  qRT-PCR results showed that the miR-193b expressions of miR-193b mimic (25, 50 and 100 pmol) groups and control group in HepG2.2.15 cells were 1.05±0.09, 1.53±0.12, 2.08±0.17 and 0.49±0.12, and the difference was statistically significant (F=261.35, P<0.001). Compared with the control group, miR-193b expression significantly increased in each transfection group (P=0.036; P=0.029; P=0.022). Flow cytometry results showed the apoptosis rates of miR193b mimic (25, 50 and 100 pmol) groups and control group were (30.28±5.22)%, (53.41±6.18)%, (79.89±7.13)% and (1.02±0.13)%, and the difference was statistically significant (F=357.19, P<0.001). Compared with the control group, the apoptosis rate significantly increased in each transfection group (P=0.025; P=0.010; P=0.007). Cell scratch assay results showed that the migration distances of miR-193b mimic (25, 50 and 100 pmol) groups and control group were (27.53±1.54)mm, (19.24±2.12)mm, (13.42±1.53)mm and (34.95±1.92)mm, and the difference was statistically significant (F=408.62, P<0.001). Compared with the control group, the migration distance significantly decreased in each transfection group (P=0.032; P=0.007; P=0.006). Transwell experimental results showed that the absorbance values of miR-193b mimic (25, 50 and 100 pmol) groups and control group were 1.02±0.12, 0.59±0.13, 0.42±0.10 and 1.68±0.16, and the difference was statistically significant (F=511.68, P<0.001). Compared with the control group, the cell invasion ability significantly weakened in each transfection group (P=0.028; P=0.005; P=0.002). Western blotting results showed that the expressions of Bax, Bcl-2, MMP-9 and MMP-2 protein of miR-193b mimic (25, 50 and 100 pmol) groups and control group had statistically significant difference (F=264.38, P<0.001; F=437.19, P<0.001; F=377.46, P<0.001; F=208.79, P<0.001). Further paired comparison showed that the expressions of Bax, Bcl-2, MMP-9 and MMP-2 protein between mimics groups and control group were statistically significant (all P<0.05). Conclusion  miR-193b can induce the apoptosis and inhibit the invasion, metastasis of HBVpositive hepatocellular cells, and the mechanism may be related to the regulation of Bax, Bcl-2, MMP-9 and MMP-2 protein expression.

Key words: Hepatitis B virus, Liver neoplasms, MicroRNAs, Tumor infiltrating