Wei Qinjun, He Wei, Lu Yajie, Yao Jun, Cao Xin
Department of Biotechnology, Nanjing Medical University, Nanjing 210029, P.R. China.
Oncol Lett. 2012 Sep;4(3):438-442. doi: 10.3892/ol.2012.744. Epub 2012 Jun 7.
Inhibitor of growth 4 (ING4) is a member of the ING family and acts as a tumor suppressor protein. To investigate the impact of ING4 on breast cancer proliferation, the present study examined the antitumor effects caused by upregulation in the expression of ING4 and its possible mechanism of effect in MCF-7 cells. A plasmid-based expression system encoding the ING4 gene was used to construct a stable cell line and overexpress ING4 in MCF-7 cells. Real-time PCR and western blot analysis were used to detect the mRNA and protein expression levels of ING4, respectively. Cell growth was examined by methylthiazolyltetrazolium (MTT) assay. Cell cycle distribution and cell apoptosis were measured by flow cytometry. The expression of p21, p53 and bax genes were tested by real-time PCR and western blot analysis. The stably transfected cell lines pcDNA3.1(+)/ING4 (with the ING4 gene) and pcDNA3.1(+) (an empty vector) were established. The expression levels of ING4 mRNA and protein in the stable cell line expressing pcDNA3.1(+)/ING4 were significantly higher than those of the two control cell lines. The cell proliferation of stably transfected cells was inhibited, and the inhibitory rate was 62.58±2.93%. Based on the changes in cell cycle distribution in stably transfected cells compared with two control cell lines, a number of cells were blocked in the G0/G1 phase 67.82±3.78% (P<0.05). In addition, the apoptotic rate was significantly higher, at 31.51±3.02% (P<0.05). Real-time PCR revealed that p21 and bax mRNA expression were increased (P<0.01), but the expression of p53 did not change significantly (P>0.05) in the stably transfected cell lines. Western blot analysis results of p21, bax and p53 were in accordance with real-time PCR results. ING4 upregulation may inhibit breast cancer cell proliferation and accelerate the process of apoptosis. It is suggested that ING4 plays a significant role in the suppression of breast cancer progression.
生长抑制因子4（ING4）是ING家族的成员，作为一种肿瘤抑制蛋白发挥作用。为了研究ING4对乳腺癌增殖的影响，本研究检测了ING4表达上调所引起的抗肿瘤作用及其在MCF-7细胞中的可能作用机制。使用基于质粒的编码ING4基因的表达系统构建稳定细胞系，并使ING4在MCF-7细胞中过表达。分别采用实时定量聚合酶链反应（Real-time PCR）和蛋白质免疫印迹法检测ING4的mRNA和蛋白表达水平。通过甲基噻唑基四氮唑（MTT）法检测细胞生长情况。采用流式细胞术检测细胞周期分布和细胞凋亡情况。通过实时定量聚合酶链反应和蛋白质免疫印迹法检测p21、p53和bax基因的表达。建立了稳定转染的细胞系pcDNA3.1(+)/ING4（含ING4基因）和pcDNA3.1(+)（空载体）。表达pcDNA3.1(+)/ING4的稳定细胞系中ING4 mRNA和蛋白的表达水平显著高于两个对照细胞系。稳定转染细胞的细胞增殖受到抑制，抑制率为62.58±2.93%。与两个对照细胞系相比，基于稳定转染细胞中细胞周期分布的变化，大量细胞被阻滞在G0/G1期，比例为67.82±3.78%（P<0.05）。此外，凋亡率显著更高，为31.51±3.02%（P<0.05）。实时定量聚合酶链反应显示，在稳定转染的细胞系中，p21和bax mRNA表达增加（P<0.01），但p53的表达没有显著变化（P>0.05）。p21、bax和p53的蛋白质免疫印迹分析结果与实时定量聚合酶链反应结果一致。ING4表达上调可能抑制乳腺癌细胞增殖并加速凋亡进程。提示ING4在抑制乳腺癌进展中发挥重要作用。
