Original Article
Copyright ©2013 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Mar 21, 2013; 19(11): 1760-1769
Published online Mar 21, 2013. doi: 10.3748/wjg.v19.i11.1760
Effects of small interfering RNA inhibit Class I phosphoinositide 3-kinase on human gastric cancer cells
Bao-Song Zhu, Li-Yan Yu, Kui Zhao, Yong-You Wu, Xiao-Li Cheng, Yong Wu, Feng-Yun Zhong, Wei Gong, Qiang Chen, Chun-Gen Xing
Bao-Song Zhu, Li-Yan Yu, Kui Zhao, Yong-You Wu, Yong Wu, Feng-Yun Zhong, Wei Gong, Qiang Chen, Chun-Gen Xing, Department of General Surgery, The Second Affiliated Hospital, Soochow University, Suzhou 215004, Jiangsu Province, China
Xiao-Li Cheng, Department of Gastroenterology, Qianfoshan Hospital, Shandong University, Jinan 250014, Shandong Province, China
Author contributions: Xing CG and Zhu BS designed the research; Xing CG, Zhu BS, and Zhao K wrote the paper; Zhao K and Yu LY collected and analyzed data; Zhu BS, Wu YY, Zhong FY, and Cheng XL selected the color figures in the paper; all authors contributed to the intellectual context and approved the final version of the manuscript.
Supported by The Natural Science Foundation of China, No. 81172348; Suzhou High-Level Talents Project, 2008-11; Suzhou Science and Technology Development Foundation, 2010SYS201031; and the Science, Education, and Health Foundation of Suzhou City, SWKQ0914 and SWKQ0916
Correspondence to: Chun-Gen Xing, Professor, Department of General Surgery, The Second Affiliated Hospital of Soochow University, 1055 San Xiang Road, Suzhou 215004, Jiangsu Province, China. xingcg@126.com
Telephone: +86-512-67784106 Fax: +86-512-67784106
Received: April 5, 2012
Revised: September 19, 2012
Accepted: December 25, 2012
Published online: March 21, 2013
Abstract

AIM: To investigate the effects of small interfering RNA (siRNA)-mediated inhibition of Class I phosphoinositide 3-kinase (Class I PI3K) signal transduction on the proliferation, apoptosis, and autophagy of gastric cancer SGC7901 and MGC803 cells.

METHODS: We constructed the recombinant replication adenovirus PI3K(I)-RNA interference (RNAi)-green fluorescent protein (GFP) and control adenovirus NC-RNAi-GFP, and infected it into human gastric cancer cells. MTT assay was used to determine the growth rate of the gastric cancer cells. Activation of autophagy was monitored with monodansylcadaverine (MDC) staining after adenovirus PI3K(I)-RNAi-GFP and control adenovirus NC-RNAi-GFP treatment. Immunofluorescence staining was used to detect the expression of microtubule-associated protein 1 light chain 3 (LC3). Mitochondrial membrane potential was measured using the fluorescent probe JC-1. The expression of autophagy was monitored with MDC, LC3 staining, and transmission electron microscopy. Western blotting was used to detect p53, Beclin-1, Bcl-2, and LC3 protein expression in the culture supernatant.

RESULTS: The viability of gastric cancer cells was inhibited after siRNA targeting to the Class I PI3K blocked Class I PI3K signal pathway. MTT assays revealed that, after SGC7901 cancer cells were treated with adenovirus PI3K(I)-RNAi-GFP, the rate of inhibition reached 27.48% ± 2.71% at 24 h, 41.92% ± 2.02% at 48 h, and 50.85% ± 0.91% at 72 h. After MGC803 cancer cells were treated with adenovirus PI3K(I)-RNAi-GFP, the rate of inhibition reached 24.39% ± 0.93% at 24 h, 47.00% ± 0.87% at 48 h, and 70.30% ± 0.86% at 72 h (P < 0.05 compared to control group). It was determined that when 50 MOI, the transfection efficiency was 95% ± 2.4%. Adenovirus PI3K(I)-RNAi-GFP (50 MOI) induced mitochondrial dysfunction and activated cell apoptosis in SGC7901 cells, and the results described here prove that RNAi of Class I PI3K induced apoptosis in SGC7901 cells. The results showed that adenovirus PI3K(I)-RNAi-GFP transfection induced punctate distribution of LC3 immunoreactivity, indicating increased formation of autophagosomes. The results showed that the basal level of Beclin-1 and LC3 protein in SGC7901 cells was low. After incubating with adenovirus PI3K(I)-RNAi-GFP (50 MOI), Beclin-1, LC3, and p53 protein expression was significantly increased from 24 to 72 h. We also found that Bcl-2 protein expression down-regulated with the treatment of adenovirus PI3K(I)-RNAi-GFP (50 MOI). A number of isolated membranes, possibly derived from ribosome-free endoplasmic reticulum, were seen. These isolated membranes were elongated and curved to engulf a cytoplasmic fraction and organelles. We used transmission electron microscopy to identify ultrastructural changes in SGC7901 cells after adenovirus PI3K(I)-RNAi-GFP (50 MOI) treatment. Control cells showed a round shape and contained normal-looking organelles, nucleus, and chromatin, while adenovirus PI3K(I)-RNAi-GFP (50 MOI)-treated cells exhibited the typical signs of autophagy.

CONCLUSION: After the Class I PI3K signaling pathway has been blocked by siRNA, the proliferation of cells was inhibited and the apoptosis of gastric cancer cells was enhanced.

Keywords: Gastric cancer cells; Class I phosphoinositide 3-kinase; RNA interference; Apoptosis; Autophagy