[Construction of eukaryotic expression vector of siRNA specific for MAD2 and its effect on the growth of gastric cell line SGC7901].
OBJECTIVE
To construct the RNA interference eukaryotic expression vector specific for human MAD2 gene and to observe its effect on the growth of gastric cancer cell line SGC7901.


METHODS
The expression vectors of pSilencer3.1/MAD2-siRNA1 and pSilencer3.1/MAD2-siRNA2 were constructed by gene recombination and then were stably transfected into the gastric carcinoma cell line SGC7901 by liposome mediation. The expression of MAD2 on the levels of protein and mRNA was detected by Western blot and RT-PCR, and the monoclone with the highest inhibition efficiency was selected. The growth of the transfected cells was assessed by MTT. And the cells treated with 1.0 mg/L vincristine (VCR) for 24 h were analyzed by FCM for cell cycle.


RESULTS
Sequence-specific siRNAs targeting MAD2 significantly down regulated the expression of MAD2 in SGC7901 cells. In MAD2-siRNA transfected cells, the rate of cell growth increased markedly and cell cycle couldn't be arrested in M phase induced by VCR, while the cells transfected with the mock vector could.


CONCLUSIONS
Down regulation of MAD2 expression of SGC7901 bv sequence-specific siRNA could accelerate the cell growth and impair the mitosis arrest of SGC7901 induced by VCR.
Du
Yu-lei
YL
Department of Gastroenterology, State Key Laboratory of Cancer Biology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Yin
Fang
F
Yang
Gui-tao
GT
Xie
Hua-hong
HH
Song
Jiu-gang
JG
Liu
Jie
J
Fan
Dai-ming
DM
D000818:Animals; D002135:Calcium-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D002277:Carcinoma / Q000473:pathology; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D005057:Eukaryotic Cells; D020868:Gene Silencing / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D064246:Mad2 Proteins; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D012097:Repressor Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D013274:Stomach Neoplasms / Q000473:pathology*
D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D034741:RNA, Small Interfering; D012097:Repressor Proteins