Chen Ying, Cao Yong, Yang Danlei, Li Kaiyan, Wang Zhengyun, Zhu Jing, Bunjhoo Hansvin, Xiong Shengdao, Xu Yongjian, Xiong Weining
Department of Respiratory Diseases, Tongji Hospital, Key Lab of Pulmonary Diseases of Health Ministry, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, P.R. China.
Exp Ther Med. 2012 Feb;3(2):255-260. doi: 10.3892/etm.2011.381. Epub 2011 Nov 16.
Overexpression of Cyclin D1 and Bcl-xL proteins has often been found in non-small-cell lung cancer (NSCLC). These two genes may play a significant role in tumorigenesis. However, the combined inhibition of the two genes in vitro is unclear in NSCLC. In this study, the effect of a combined intervention on Cyclin D1 and Bcl-xL in NSCLC is assessed and discussed. Three recombinant plasmids that expressed a cytomegalovirus (CMV) promoter-driven micro30 short hairpin RNA (shRNA) targeting the Cyclin D1 gene (Cyclin D1 shRNA), the Bcl-xL gene (Bcl-xL shRNA) and a combination of the two genes (Cyclin D1-Bcl-xL shRNA), based on the plasmid pcDNA6.2-GW/EmGFP-miR, were constructed. The cell lines A549 and NCI-H441 were divided into four groups; blank control (untreated cells), Cyclin D1 shRNA, Bcl-xL shRNA and Cyclin D1-Bcl-xL shRNA (transfected cells), respectively. The expression of mRNA and protein of Cyclin D1 or Bcl-xL was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. The apoptosis and proliferation of the two cell lines were evaluated by dimethylthiazol-diphenyltetrazolium bromide (MTT), cell count and flow cytometry. The recombinant plasmid sufficiently mediated the RNA interference (RNAi) effects in A549 and NCI-H441 cells. The expression levels of mRNA and protein of Cyclin D1 or Bcl-xL in the three intervention groups were significantly reduced compared to the untreated cells (P<0.05). No statistical differences were found among the combined shRNAs and single shRNA regarding Cyclin D1 or Bcl-xL, respectively (P>0.05). In the assessment of proliferation and apoptosis, it was found that in all three intervention groups there was significant inhibition of cell proliferation and promotion of cell apoptosis compared with the untreated cells (P<0.05). Furthermore, the combined interference of the two genes was more effective than either single interference (P<0.05). Our results suggested that the combined targeting of Cyclin D1 and Bcl-xL genes has potential for NSCLC investigation, providing increased efficacy over Cyclin D1 or Bcl-xL inhibition alone.
在非小细胞肺癌（NSCLC）中，经常发现细胞周期蛋白D1（Cyclin D1）和Bcl-xL蛋白过表达。这两个基因可能在肿瘤发生中起重要作用。然而，在NSCLC中，这两个基因在体外的联合抑制作用尚不清楚。在本研究中，评估并讨论了联合干预对NSCLC中Cyclin D1和Bcl-xL的影响。基于质粒pcDNA6.2-GW/EmGFP-miR构建了三种重组质粒，分别表达靶向Cyclin D1基因（Cyclin D1 shRNA）、Bcl-xL基因（Bcl-xL shRNA）以及这两个基因组合（Cyclin D1-Bcl-xL shRNA）的巨细胞病毒（CMV）启动子驱动的micro30短发夹RNA（shRNA）。将A549和NCI-H441细胞系分为四组，分别为空白对照（未处理细胞）、Cyclin D1 shRNA组、Bcl-xL shRNA组和Cyclin D1-Bcl-xL shRNA组（转染细胞）。分别通过逆转录-聚合酶链反应（RT-PCR）和蛋白质印迹法检测Cyclin D1或Bcl-xL的mRNA和蛋白表达。通过噻唑蓝（MTT）、细胞计数和流式细胞术评估这两种细胞系的凋亡和增殖情况。重组质粒在A549和NCI-H441细胞中充分介导了RNA干扰（RNAi）效应。与未处理细胞相比，三个干预组中Cyclin D1或Bcl-xL的mRNA和蛋白表达水平均显著降低（P<0.05）。在Cyclin D1或Bcl-xL方面，联合shRNAs与单一shRNA之间分别未发现统计学差异（P>0.05）。在增殖和凋亡评估中发现，与未处理细胞相比，所有三个干预组的细胞增殖均受到显著抑制，细胞凋亡均得到促进（P<0.05）。此外，两个基因的联合干扰比单一干扰更有效（P<0.05）。我们的结果表明，联合靶向Cyclin D1和Bcl-xL基因在NSCLC研究中具有潜力，比单独抑制Cyclin D1或Bcl-xL具有更高的疗效。
