国际肿瘤学杂志

国际肿瘤学杂志 ›› 2017, Vol. 44 ›› Issue (1): 1-5.doi: 10.3760/cma.j.issn.1673-422X.2017.01.001

• 论著 •    下一篇

Pyk2基因对肝癌Hep3B细胞增殖、侵袭和迁移的影响

左凯,薛栋,孔丽,谢琳琳,李文玉,燕小辉,夏修良   

  1. 256610 山东省滨州市人民医院感染二科（左凯、谢琳琳、李文玉、燕小辉），普通外科（薛栋、夏修良），静配中心（孔丽）
  • 出版日期:2017-01-08 发布日期:2016-12-07
  • 通讯作者: 薛栋，Email: doctorxuedong@163.com E-mail:doctorxuedong@163.com
  • 基金资助:

    滨州市科技发展计划（2014ZC0128）

Targeting Pyk2 gene on the proliferation, invasion and migration induction of hepatocelluar cancer Hep3B cells

 Zuo Kai, Xue Dong, Kong Li, Xie Linlin, Li Wenyu, Yan Xiaohui, Xia Xiuliang   

  1. Second Department of Infectory Diseases, People′s Hospital of  Binzhou City, Shandong Province, Binzhou 256610, China
  • Online:2017-01-08 Published:2016-12-07
  • Contact: Xue Dong, Email: doctorxuedong@163.com E-mail:doctorxuedong@163.com
  • Supported by:

    Science and Technology Development Plans of Binzhou City of Shandong Province, China（2014ZC0128）

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摘要： 目的探讨沉默富含脯氨酸的酪氨酸激酶2(Pyk2)基因对人肝癌Hep3B细胞增殖、侵袭及迁移的影响。方法构建Pyk2基因RNA干扰（RNAi）载体，脂质体转染至肝癌Hep3B细胞，实验分3组，Pyk2 RNA干扰组(siRNA组)：转染携带Pyk2基因RNAi的质粒；阴性对照组：转染不携带Pyk2基因RNAi的空载质粒；空白对照组：未转染任何质粒。应用反转录聚合酶链反应(RT-PCR)及Western blotting鉴定Pyk2基因和蛋白表达；采用四甲基偶氮唑蓝（MTT）、Transwell侵袭和划痕实验分别检测细胞增殖、侵袭及迁移等生物学特性。结果Pyk2基因干扰后肝癌Hep3B细胞Pyk2 mRNA的表达（0.16±0.03）较阴性对照组（0.74±0.13）和空白对照组（0.77±0.16）均明显下降，差异均有统计学意义（t=51.46，P=0.000；t=53.21，P=0.000）。Pyk2基因干扰后肝癌Hep3B细胞Pyk2蛋白的表达（0.24±0.06）较阴性对照组（0.83±0.05）和空白对照组（0.91±0.06）均明显下降，差异均有统计学意义（t=57.29，P=0.000；t=68.53，P=0.000）。siRNA组48 h细胞增殖抑制率（26.17%±0.28%）较阴性对照组（9.28%±0.22%）和空白对照组（6.47%±0.31%）明显提高，差异均有统计学意义（t=31.45，P=0.004；t=34.64，P=0.002）。siRNA组细胞的穿膜细胞数（32.5±8.5）/10 HP明显低于阴性对照组（98.4±12.3）/10 HP和空白对照组（112.6±11.3）/10 HP，差异有统计学意义（t=95.64，P=0.000；t=105.17，P=0.000）。siRNA组的划痕愈合率（28.17%±1.46%）显著低于阴性对照组（77.38%±2.24%）和空白对照组（79.41%±3.17%），差异有统计学意义（t=85.86，P=0.000；t=89.37，P=0.000)。结论Pyk2基因参与肝癌Hep3B细胞的增殖、侵袭及迁移，与肿瘤发展、侵袭、转移等密切相关，Pyk2有望成为肝癌诊断和治疗的分子靶标。

关键词: 肝肿瘤, 细胞增殖, 肿瘤浸润, RNA干扰, 富含脯氨酸的酪氨酸激酶2

Abstract: ObjectiveTo investigate the influence of prolinerich tyrosine kinase 2 (Pyk2) gene RNA interference on proliferation, invasion and migration of Hep3B hepatocellular carcinoma cells. MethodsThe Pyk2 gene RNA interference vector was transfected in Hep3B hepatocellular carcinoma cells by lipofectamine. The Hep3B cells divided into three groups: siRNA group (the vector with Pyk2 RNAi gene was transfected), negative control group (the vector without Pyk2 RNAi gene was transfected), and blank control group (no vectors was transfected). Pyk2 mRNA and protein were detected using reverse transcription reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. The biological behavior including cell proliferation, invasion and migration were detected by 3-(4, 5-dimethyl-2-thiazoly)-2, 5-diphenyl-2H-tetrazolium bromide (MTT), transwell and wound healing assay, respectively. ResultsThe expression of Pyk2 mRNA of Hep3B cell line in siRNA group (0.16±0.03) was significantly decreased than those in negative group (0.74±0.13) and blank control group (0.77±0.16), with statistically significant differences (t=51.46, P=0.000; t=53.21, P=0.000). The expression of Pyk2 protein of Hep3B cell line in siRNA group (0.24±0.06) was significantly decreased than those in negative group (0.83±0.05) and blank control group (0.91±0.06), with statistically significant differences (t=57.29, P=0.000; t=68.53, P=0.000). The cell proliferation inhibition rate at 48 hours in siRNA group (26.17%±0.28%) was significantly raised than those in negative group (9.28%±0.22%) and blank control group (6.47%±0.31%), with statistically significant differences (t=31.45, P=0.004; t=34.64, P=0.002). The number of transmembrane cells in siRNA group (32.5±8.5)/10 HP was significantly declined than those in negative group (98.4±12.3)/10 HP and blank control group (112.6±11.3)/10 HP, with statistically significant differences (t=95.64, P=0.000; t=105.17, P=0.000). The wound healing assay in siRNA group (28.17%±1.46%) was significantly lower than those in negative group (77.38%±2.24%) and blank control group (79.41%±3.17%), with statistically significant (t=85.86, P=0.000; t=89.37, P=0.000). ConclusionPyk2 gene involves the proliferation, invasion and migration of Hep3B cells, which has close correction with development and metastasis of hepatocellular carcinoma. Pyk2 gene is very helpful to become a molecular target for the diagnosis and treatment of hepatocellular carcinoma.

Key words: Liver neoplasms, Cell proliferation, Neoplasm invasiveness, RNA interference, Proline-rich tyrosine kinase 2