Sun Hua-Wen, Tong Shi-Lun, He Jie, Wang Qi, Zou Li, Ma Shu-Jing, Tan Hai-Yan, Luo Jian-Fei, Wu Hong-Xue
Department of Gastrointestinal Surgery, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China.
World J Gastroenterol. 2007 Jul 7;13(25):3517-22. doi: 10.3748/wjg.v13.i25.3517.
To evaluate the effects of adenovirus-mediated gene transfer of RhoA siRNA and RhoC siRNA on proliferation and invasion of SGC7901 cells by Rho/PI3K/Akt pathway.
Plasmid of RhoA siRNA and RhoC siRNA were constructed and transfected into SGC7901 cells. siRNA and LY294002 (PI3K inhibitor) were designed as the control group. The mRNA and protein expressions of RhoA and RhoC were respectively detected with RT-PCR and western blotting. In order to find out the changes of proliferation and invasion power of SGC7901 cell lines, we analyzed the data by MTT, Boyden chamber and evaluated apoptosis of cell with flow cytometry. We treated BALB/C nude mice with RhoA and RhoC-siRNA, and tumor control rate (%) in nude mice was calculated.
RhoA and RhoC siRNA transfections specifically down-regulated the corresponding mRNA and protein levels in SGC7901 Cells. The experiment of permeated artificial basal membrane showed that the invasion power of SGC7901 cell lines are on the decline after treatment of Ad-RhoA and RhoC-siRNA (12.64 +/-3.27 vs 87.38 +/- 17.38, P < 0.05). The values of 490 nm wavelength light absorption were different in the five groups. The number of alive cells in the group of RhoA and RhoC-siRNA was lower than others in the 6(th) d (0.71 +/- 0.01 vs 3.82 +/- 0,11 P < 0.05). The apoptosis rate of transfected RhoA and RhoC-siRNA group with FACS were 19.07% +/- 1.78 and there were significant differences between treated and control groups (19.07 +/- 1.78% vs 1.23 +/- 0.11%, P < 0.01). The tumor transplantation experiment in BALB/C nude mice showed intratumoral injection of RhoA or RhoC siRNA can inhibit tumor growth.
RhoA and RhoC siRNA gene therapy mediated by adenovirus may be useful for inhibiting growth and invasion of SGC7901 through a PI3K/Akt pathway. These results provide a novel therapeutic target in preventing gastric cancer cell invasion and metastasis.
通过Rho/PI3K/Akt信号通路评估腺病毒介导的RhoA小干扰RNA（siRNA）和RhoC siRNA基因转染对SGC7901细胞增殖和侵袭的影响。
构建RhoA siRNA和RhoC siRNA质粒并转染至SGC7901细胞。将siRNA和LY294002（PI3K抑制剂）设为对照组。采用逆转录聚合酶链反应（RT-PCR）和蛋白质印迹法分别检测RhoA和RhoC的信使核糖核酸（mRNA）及蛋白表达。为了解SGC7901细胞系增殖和侵袭能力的变化，通过噻唑蓝（MTT）比色法、Boyden小室法进行数据分析，并用流式细胞术评估细胞凋亡情况。用RhoA和RhoC-siRNA处理BALB/C裸鼠，计算裸鼠的肿瘤抑制率（%）。
RhoA和RhoC siRNA转染可特异性下调SGC7901细胞中相应的mRNA和蛋白水平。穿膜人工基底膜实验显示，经腺病毒载体RhoA和RhoC-siRNA处理后，SGC7901细胞系的侵袭能力下降（12.64±3.27对87.38±17.38，P<0.05）。五组在490nm波长处的吸光度值不同。在第6天，RhoA和RhoC-siRNA组的活细胞数低于其他组（0.71±0.01对3.82±0.11，P<0.05）。经流式细胞术检测，转染RhoA和RhoC-siRNA组的凋亡率为19.07%±1.78，处理组与对照组之间有显著差异（19.07±1.78%对1.23±0.11%，P<0.01）。BALB/C裸鼠肿瘤移植实验表明，瘤内注射RhoA或RhoC siRNA可抑制肿瘤生长。
腺病毒介导的RhoA和RhoC siRNA基因治疗可能通过PI3K/Akt信号通路抑制SGC7901细胞的生长和侵袭。这些结果为预防胃癌细胞侵袭和转移提供了新的治疗靶点。
