Expression of livin in gastric cancer and induction of apoptosis in SGC-7901 cells by shRNA-mediated silencing of livin gene

• Published in: Biomedicine & pharmacotherapy Vol. 64; no. 5; pp. 333 - 338
• Main Authors: Wang, T.-S, Ding, Q.-Q, Guo, R.-H, Shen, H, Sun, J, Lu, K.-H, You, S.-H, Ge, H.-M, Shu, Y.-Q, Liu, P
• Format: Journal Article
• Language: English
• Published: Paris Elsevier SAS 01.05.2010; Elsevier
• Subjects: Adaptor Proteins, Signal Transducing - genetics; Adaptor Proteins, Signal Transducing - metabolism; Adenocarcinoma - genetics; Adult; Aged; Antineoplastic Agents - administration & dosage; Antineoplastic Agents - pharmacology; Apoptosis; Apoptosis - genetics; Biological and medical sciences; Blotting, Western; Cell Line, Tumor; Cisplatin - administration & dosage; Cisplatin - pharmacology; Female; Flow Cytometry; Fluorouracil - administration & dosage; Fluorouracil - pharmacology; Gastroenterology. Liver. Pancreas. Abdomen; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Inhibitor of Apoptosis Proteins - genetics; Inhibitor of Apoptosis Proteins - metabolism; Inhibitory Concentration 50; Internal Medicine; Livin; Lymphatic Metastasis; Male; Medical Education; Medical sciences; Middle Aged; Neoplasm Proteins - genetics; Neoplasm Proteins - metabolism; Pharmacology. Drug treatments; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - metabolism; RNA, Small Interfering - genetics; RNAi; RT-PCR; Stomach neoplasms; Stomach Neoplasms - genetics; Stomach. Duodenum. Small intestine. Colon. Rectum. Anus; Tumors; RNAi; RT-PCR; Stomach neoplasms; Livin; Apoptosis; RNA interference; RNA-directed DNA polymerase; Stomach cancer; Gene; Genetics; Reverse transcription polymerase chain reaction; Gastric disease; Enzyme; Induction; Transferases; Malignant tumor; In vitro; Gene silencing; Nucleotidyltransferases; Apoptosis inhibitory protein; Cell death; Digestive diseases; Molecular biology; Short hairpin RNA; Cancer
• ISSN: 0753-3322; 1950-6007
• DOI: 10.1016/j.biopha.2009.06.002

Abstract Background Because of increased resistance to apoptosis in tumor cells, inhibition of specific anti-apoptotic factors may provide a rational approach for the development of novel therapeutic strategies. Livin, a novel inhibitor of apoptosis protein family, has been found to be expressed in various malignancies and is suggested to have poorly prognostic significance. However, no data are available concerning the significance of livin in gastric cancer. In this study, we detected the expression of livin in human gastric carcinoma and investigated the apoptotic susceptibility of SGC – 7901 cell by shRNA-mediated silencing of the livin gene. Methods The mRNA and protein expression of livin were analyzed by RT-PCR and western blot assay. The relationship between livin expression and clinical pathologic parameters was investigated. The small interfering RNA eukaryotic expression vector specific to livin was constructed by gene recombination, and the nucleic acid was sequenced. Then it was transfected into SGC-7901 cells by Lipofectamin 2000. RT-PCR and Western blot assay were used to validate gene-silencing efficiency of livin in SGC-7901 cells. Stable clones were obtained by G418 screening. The cell apoptosis was assessed by flow cytometry (FCM). Cell growth state and 50 % inhibition concentration (IC50) of 5-FU and cisplatin was determined by MTT method. Results The expression of livin mRNA and protein were detected in 19 of 40 gastric carcinoma cases (47.5%) and SGC-7901 cells. No expression of livin was detected in tumor adjacent tissues and benign gastric lesion. The positive correlation was found between livin expression and poor differentiation of tumors as well as lymph node metastases ( P < 0.05). Four small interfering RNA eukaryotic expression vector specific to livin were constructed by gene recombination. And one of them can efficiently decrease the expression of livin, the inhibition of the gene was not less than 70% ( P < 0.01). The recombinated plasmids were extracted and transfected gastric cancer cells. The stable clones were obtained by G418 screening, and were amplified and cultured. When livin gene was silenced, the reproductive activity of the gastric cancer cells was significantly lower than the control groups( P < 0.05). The study also showed that IC50 of 5-Fu and cisplatin on gastric cancer cells treated by shRNA was decreased and the cells were more susceptible to proapoptotic stimuli (5-Fu and cisplatin) ( P < 0.01). Conclusions Livin is overexpressed in gastric carcinoma with a relationship to tumor differentiation and lymph node metastases, which is suggested to be one of the molecular prognostic factors for some cases of gastric cancer. ShRNA can inhibit livin expression in SGC-7901 cells and induce cell apoptosis. Livin may serve as a new target for apoptosis-inducing therapy of gastric cancer.