Expression of livin in gastric cancer and induction of apoptosis in SGC-7901 cells by shRNA-mediated silencing of livin gene.
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.
Wang
T-S
TS
Department of Oncology, the First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, Jiangsu Province, China.
Ding
Q-Q
QQ
Guo
R-H
RH
Shen
H
H
Sun
J
J
Lu
K-H
KH
You
S-H
SH
Ge
H-M
HM
Shu
Y-Q
YQ
Liu
P
P
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000378:metabolism; D000230:Adenocarcinoma / Q000235:genetics; D000328:Adult; D000368:Aged; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology; D017209:Apoptosis / Q000235:genetics; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000008:administration & dosage / Q000494:pharmacology; D005260:Female; D005434:Flow Cytometry; D005472:Fluorouracil / Q000008:administration & dosage / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics* / Q000378:metabolism; D020128:Inhibitory Concentration 50; D008207:Lymphatic Metastasis; D008297:Male; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000235:genetics*
D048868:Adaptor Proteins, Signal Transducing; D000970:Antineoplastic Agents; C419866:BIRC7 protein, human; D051034:Inhibitor of Apoptosis Proteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D002945:Cisplatin; D005472:Fluorouracil