Liu Hong-Chun, Zhang Gen-Hao, Liu Yu-Han, Wang Pan, Ma Jun-Fen, Su Li-Sha, Li Sheng-Lei, Zhang Lan, Liu Jun-Wen
Department of Clinical Laboratory, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China.
Department of Clinical Laboratory, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China.
Biomed Pharmacother. 2014 Sep;68(7):833-9. doi: 10.1016/j.biopha.2014.08.008. Epub 2014 Aug 18.
Observe how specific small RNA interference (siRNA) aimed at TPX2 gene suppresses TPX2 gene expression in esophageal cancer EC9706 cells and the effect on esophageal cancer cell growth and invasion ability.
Transfect TPX2 siRNA into EC9706 cells via lipofectamin 2000. The experiments were divided into three groups, a negative control, a blank control and an siRNA interference group (24h, 48h, 72h, 96h). We examined RNA and protein level alteration of the TPX2 gene after TPX2 siRNA transfection by RT-PCR and Western blot analysis. Detection of how TPX2 siRNA influences EC9706 cell proliferation was done by MTT, cell apoptosis monitored through Tunel assay, in vitro invasion ability via Boyden chamber and cell cycle change by flow cytometry.
After effective siRNA transfection, TPX2 mRNA and protein expression level in siRNA interference group were (0.31±0.08, 0.39±0.12),72h after transfection, significantly lower than blank control group (1.00±0.01) and negative control group (0.98±0.11), (F=71.182, t1=8.17, t2=7.90, P<0.05); MTT results demonstrated that cell growth and proliferation were inhibited and the inhibition rate was up to 35.4% (P<0.05) compared with the control group. TUNEL results indicated that cell apoptosis index in siRNA interference group was 18.28±0.35, higher than that in blank control group (4.07±0.26)and negative control group (4.13±0.22), (F=244.5, t1=60.61, t2=53.32, P<0.01). Boyden chamber results showed that the transmembrane cell number was 45.30±8.08 in siRNA interference group, less than blank control group (121.90±7.83), (F=122.46, t1=11.81, t2=10.47, P<0.01); besides, in siRNA interference group cell invasion inhibition rate was 71.42±9.12, higher than negative control group (5.65±3.55), (t=14.256, P<0.01). Flow cytometry results illustrated that more EC9706 cells went into apoptosis and cell cycle arrested in S phase. Similar results were obtained by in vivo transplantation, as TPX2 siRNA transfection significantly reduced tumor growth of the xenograft in nude mice.
siRNA could effectively inhibit the invasion and metastasis of EC9706 cells, promote the apoptosis of tumor cells and may become a new approach for treatment of esophageal carcinoma.
观察针对TPX2基因的特异性小RNA干扰（siRNA）对食管癌EC9706细胞中TPX2基因表达的抑制作用及其对食管癌细胞生长和侵袭能力的影响。
通过脂质体2000将TPX2 siRNA转染至EC9706细胞。实验分为三组，即阴性对照组、空白对照组和siRNA干扰组（24小时、48小时、72小时、96小时）。采用RT-PCR和蛋白质免疫印迹分析检测TPX2 siRNA转染后TPX2基因的RNA和蛋白质水平变化。采用MTT法检测TPX2 siRNA对EC9706细胞增殖的影响，通过Tunel法监测细胞凋亡，利用Boyden小室检测体外侵袭能力，采用流式细胞术检测细胞周期变化。
有效转染siRNA后，siRNA干扰组转染72小时后TPX2 mRNA和蛋白质表达水平分别为（0.31±0.08，0.39±0.12），显著低于空白对照组（1.00±0.01）和阴性对照组（0.98±0.11），（F=71.182，t1=8.17，t2=7.90，P<0.05）；MTT结果显示细胞生长和增殖受到抑制，与对照组相比抑制率高达35.4%（P<0.05）。Tunel结果表明，siRNA干扰组细胞凋亡指数为18.28±0.35，高于空白对照组（4.07±0.26）和阴性对照组（4.13±0.22），（F=244.5，t1=60.61，t2=53.32，P<0.01）。Boyden小室结果显示，siRNA干扰组穿膜细胞数为45.30±8.08，少于空白对照组（121.90±7.83），（F=122.46，t1=11.81，t2=10.47，P<0.01）；此外，siRNA干扰组细胞侵袭抑制率为71.42±9.12，高于阴性对照组（5.65±3.55），（t=14.256，P<0.01）。流式细胞术结果表明，更多的EC9706细胞进入凋亡状态，细胞周期停滞于S期。体内移植实验获得了类似结果，即TPX2 siRNA转染显著降低了裸鼠异种移植瘤的生长。
siRNA可有效抑制EC9706细胞的侵袭和转移，促进肿瘤细胞凋亡，有望成为食管癌治疗的新方法。
