国际肿瘤学杂志

国际肿瘤学杂志 ›› 2021, Vol. 48 ›› Issue (1): 18-23.doi: 10.3760/cma.j.cn371439-20200310-00003

• 论著 • 上一篇    下一篇

靶向沉默PRL-3基因对肺癌细胞增殖、迁移、侵袭及上皮间质转化的影响

王宁菊1, 陈冬梅2, 章恒3, 胡萍1, 王燕1()   

  1. 1宁夏医科大学总医院肿瘤内科,银川 750004
    2安徽省胸科医院肿瘤一科,合肥230022
    3安徽省胸科医院心胸外科,合肥 230022
  • 收稿日期:2020-03-10 修回日期:2020-10-02 出版日期:2021-01-08 发布日期:2021-01-21
  • 通讯作者: 王燕 E-mail:mgwy1974@163.com
  • 基金资助:
    宁夏自然科学基金(NZ17143);银川市科学技术局科技攻关项目

Effects of targeted silencing of PRL-3 gene on proliferation, migration, invasion and epithelial-mesenchymal transition of lung cancer cells

Wang Ningju1, Chen Dongmei2, Zhang Heng3, Hu Ping1, Wang Yan1()   

  1. 1Department of Medical Oncology, General Hospital of Ningxia Medical University, Yinchuan 750004, China
    2First Department of Oncology, Anhui Chest Hospital, Hefei 230022, China
    3Department of Cardiothoracic Surgery, Anhui Chest Hospital, Hefei 230022, China
  • Received:2020-03-10 Revised:2020-10-02 Online:2021-01-08 Published:2021-01-21
  • Contact: Wang Yan E-mail:mgwy1974@163.com
  • Supported by:
    Natural Science Foundation of Ningxia(NZ17143);Science and Technology Project of Yinchuan Science and Technology Bureau

RichHTML

35

PDF

239

摘要：

目的 观察慢病毒介导的shRNA沉默肝再生磷酸酶-3(PRL-3)基因对肺癌A549细胞增殖、迁移、侵袭的影响及其对上皮间质转化(EMT)信号通路的调控。方法 将携带PRL-3 shRNA的慢病毒载体转染肺癌A549细胞,建立稳定沉默PRL-3的肺癌细胞株。将细胞分为空白对照组、NC shRNA组(阴性对照组)和PRL-3 shRNA组(PRL-3抑制RNAi慢病毒组)。CCK-8法、平板克隆形成实验、Transwell和侵袭小室实验分别检测各组细胞增殖、迁移和侵袭能力。实时荧光定量PCR检测转染后上皮钙黏素(E-cadherin)、Snail mRNA的相对表达水平。结果 稳定沉默PRL-3的细胞株构建成功,PRL-3 shRNA组PRL-3基因敲减效率达到83.5%。CCK-8法检测A549细胞增殖能力,空白对照组、NC shRNA组和PRL-3 shRNA组24 h吸光度(A)值分别为0.296±0.008、0.342±0.007、0.292±0.004,差异具有统计学意义(F=106.300,P<0.001),PRL-3 shRNA组明显低于NC shRNA组(P<0.001);转染后48、72、96 h,PRL-3 shRNA组细胞增殖能力也明显受到抑制。克隆形成实验结果显示,空白对照组、NC shRNA组、PRL-3 shRNA组细胞集落形成数分别为(166.7±6.7)个、(158.0±6.1)个、(119.7±1.5)个(F=67.290,P<0.001),PRL-3 shRNA组细胞克隆形成能力较NC shRNA组显著下降(P<0.001)。空白对照组、NC shRNA组和PRL-3 shRNA组迁移细胞数分别为(100.0±1.9)个、(98.8±1.9)个和(44.6±7.6)个(F=430.300,P<0.001),PRL-3 shRNA组细胞迁移能力明显低于NC shRNA组(P<0.001);3组侵袭细胞数分别为(117.7±4.1)个、(113.1±6.6)个和(55.6±8.4)个(F=247.200,P<0.001),PRL-3 shRNA组细胞侵袭能力明显低于NC shRNA组(P<0.001)。实时荧光定量PCR检测结果显示,沉默PRL-3表达后,A549细胞中E-cadherin mRNA相对表达水平显著上调,Snail mRNA水平显著下调。结论 沉默PRL-3表达可抑制A549细胞的增殖、迁移和侵袭能力,PRL-3可能通过EMT途径影响肺癌细胞的侵袭。

关键词: 肺肿瘤, 促肝细胞再生磷酸酶-3, 侵袭, 上皮间质转化

Abstract:

Objective To observe the effects of lentivirus-mediated shRNA silencing of PRL-3 gene on the proliferation, migration and invasion of lung cancer A549 cells and regulation of epithelial-mesenchymal transition (EMT) signaling pathway. Methods Lung cancer A549 cells were transfected with lentiviral interference vector carrying PRL-3 shRNA to build a stable PRL-3-silencing cell line. The cells were divided into blank control group, NC shRNA group (negative control group) and PRL-3 shRNA group (PRL-3 inhibiting RNAi lentivirus group). CCK-8 method, colony formation assay, Transwell and invasion chamber assay were performed to detect the proliferation, migration and invasion ability of A549 cells respectively. The expressions of E-cadherin and Snail mRNA were detected by real-time fluorescent quantitative PCR. Results The stable PRL-3-silencing cell line was successfully constructed. The knockdown efficiency of PRL-3 gene in the PRL-3 shRNA group reached 83.5%. CCK-8 method detected the proliferation ability of A549 cells, and the results showed the 24 h absorbance (A) values of A549 cells in the blank control group, NC shRNA group and PRL-3 shRNA group were 0.296±0.008, 0.342±0.007 and 0.292±0.004, with a statistically significant diffe-rence (F=106.300, P<0.001), and the PRL-3 shRNA group was significantly lower than the NC shRNA group (P<0.001); at 48, 72, 96 h after transfection, the cell proliferation abilities of the PRL-3 shRNA group were also significantly inhibited. Colony formation assay showed that the numbers of colony formation in the blank control group, NC shRNA group and PRL-3 shRNA group were 166.7± 6.7, 158.0±6.1 and 119.7±1.5 (F=67.290, P<0.001). The ability of colony formation of the PRL-3 shRNA group was significantly lower than that of the NC shRNA group (P<0.001). The numbers of migrated cells in the blank control group, NC shRNA group and PRL-3 shRNA group were 100.0±1.9, 98.8±1.9 and 44.6±7.6 (F=430.300, P<0.001). The migration ability of the PRL-3 shRNA group was significantly lower than that of the NC shRNA group (P<0.001). The numbers of invaded cells in the three groups were 117.7±4.1, 113.1±6.6 and 55.6±8.4 (F=247.200, P<0.001). The invasion ability of the PRL-3shRNA group was significantly lower than that of the NC shRNA group (P<0.001). Real-time fluorescent quantitative PCR detection results showed that after silencing the expression of PRL-3, the relative expression level of E-cadherin mRNA in A549 cells was significantly up-regulated, and the level of Snail mRNA was significantly down-regulated. Conclusion PRL-3 silencing can inhibit the proliferation, migration and invasion of A549 cells effectively. PRL-3 may affect the invasion of lung cancer cells through the EMT pathway.

Key words: Lung neoplasms, Phosphatase of regenerating liver-3, Invasion, Epithelial-mesenchymal transition