Original Article
Copyright ©2013 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Feb 28, 2013; 19(8): 1239-1246
Published online Feb 28, 2013. doi: 10.3748/wjg.v19.i8.1239
Effect of silencing of high mobility group A2 gene on gastric cancer MKN-45 cells
Chun-Hui Wei, Li-Xiu Wei, Ming-Yu Lai, Jia-Zhuang Chen, Xi-Jing Mo
Chun-Hui Wei, Li-Xiu Wei, Ming-Yu Lai, Jia-Zhuang Chen, Department of Geriatric Gastroenterology, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China
Xi-Jing Mo, Department of Gastroenterology, the First Affiliated Hospital of Guangxi Chinese Medical University, Nanning 530001, Guangxi Zhuang Autonomous Region, China
Author contributions: Wei CH and Lai MY contributed equally to this work; Wei CH, Wei LX, Lai MY, Chen JZ and Mo XJ designed the research; Lai MY was responsible for the whole study; Wei CH, Wei LX, Chen JZ and Mo XJ performed the research and analyzed the data; and Wei CH wrote the paper.
Supported by The Natural Science Foundation of Guangxi, No. 2010GXNSFA013166; and the Key Project of Health Department of Guangxi, No. Zhong 2010021
Correspondence to: Ming-Yu Lai, Professor, Department of Geriatric Gastroenterology, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China. laimingyu2005103@yahoo.com.cn
Telephone: +86-771-5356585 Fax: +86-771-5356585
Received: September 3, 2012
Revised: January 3, 2013
Accepted: January 11, 2013
Published online: February 28, 2013
Processing time: 178 Days and 16.1 Hours
Abstract

AIM: To investigate the effect of high mobility group A2 (HMGA2) gene silencing on gastric cancer MKN-45 cells in vitro.

METHODS: HMGA2 short hairpin RNA (shRNA) expression plasmids were constructed, including a pair of random scrambled sequences. Human gastric cancer cell line MKN-45 cells were divided into three groups: blank control group (non-transfected cells), transfected group (cells transfected with HMGA2 shRNA recombinant plasmid) and scrambled sequence group (transfected with random scrambled plasmid). Cells were transfected with HMGA2 shRNA recombinant plasmids and scrambled plasmid in vitro, and the cells transfection efficiency was assayed by fluorescence microscopy. The HMGA2 messenger RNA (mRNA) expression was detected by reverse transcription polymerase chain reaction, gastric cancer cells apoptosis was detected by flow cytometry, cell proliferation was detected by methyl thiazol tetrazolium, and the protein expression of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), P27, caspase-9 and B-cell leukemia/lymphoma-2 (Bcl-2) were analyzed by Western blotting.

RESULTS: Compared with the blank control group and the scrambled sequence group, the levels of HMGA2 mRNA and protein expression in the transfected group were significantly reduced (P < 0.05). The relative HMGA2 mRNA expression levels of the blank control group, transfected group and scrambled sequence group were 0.674 ± 0.129, 0.374 ± 0.048 and 0.689 ± 0.124, respectively. The relative HMGA2 protein expression levels of the blank control group, transfected group and scrambled sequence group were 0.554 ± 0.082, 0.113 ± 0.032 and 0.484 ± 0.123, respectively. Moreover, transfection with the scrambled sequence had no effect on the expression of HMGA2. After being transfected with shRNA for 24, 48 and 72 h, the cell apoptotic rates of the transfected group were 21.65% ± 0.28%, 39.98% ± 1.82% and 24.51% ± 0.93%, respectively, which significantly higher than those of blank control group (4.72% ± 1.34%, 5.83% ± 0.13% and 5.22% ± 1.07%) and scrambled sequence group (4.28% ± 1.33%, 7.87% ± 1.43% and 6.71% ± 0.92%). After 24, 48 and 72 h, the cell proliferation inhibition rates in the transfected group were 31.57% ± 1.17%, 39.45% ± 2.07% and 37.56% ± 2.32%, respectively; the most obvious cell proliferation inhibition appeared at 48 h after transfection. Compared with the blank control group and scrambled sequence group, after transfection of shRNA for 72 h, the protein expression levels of PI3K (0.042 ± 0.005 vs 0.069 ± 0.003, 0.067 ± 0.05), Akt (0.248 ± 0.004 vs 0.489 ± 0.006, 0.496 ± 0.104) and Bcl-2 (0.295 ± 0.084 vs 0.592 ± 0.072, 0.594 ± 0.109) were significantly reduced. The protein expression levels of P27 (0.151 ± 0.010 vs 0.068 ± 0.014, 0.060 ± 0.013) and caspase-9 (0.136 ± 0.042 vs 0.075 ± 0.010, 0.073 ± 0.072) were significantly upregulated.

CONCLUSION: HMGA2 shRNA gene silencing induces apoptosis and suppresses proliferation of MKN-45 cells.

Keywords: Gastric cancer cells; RNA interference; High mobility group A2; Proliferation; Apoptosis