[Construction of a eukaryotic vector expressing MASPIN gene and its effect on cell apoptosis in gastric cancer cell line SGC7901].
OBJECTIVE
MASPIN gene is closely associated with carcinogengsis and plays a key role in cell proliferation, adhesion, migration and apoptosis. This study was to construct a recombinant eukaryotic vector expressing MASPIN, and explore the effect of MASPIN overexpression on the apoptosis in human gastric carcinoma cell line SGC7901.


METHODS
A eukaryotic expression vector MASIPIN/PCR2.1 was constructed and transfected into SGC7901 cells. RT-PCR and Western blot were used to detect the expression changes of MASPIN and Bax/Bcl-2. TNF-related apoptosis inducing ligand (TRAIL) (50ng/ml) was used to induce apoptosis at different time courses. DNA apoptotic ladders were determined using agarose gel electrophoresis (AGE). Cell apoptosis was measured by flow cytometry (FCM).


RESULTS
Recombinant plasmid MASIPIN/PCR2.1 was successfully constructed and transfected into SGC7901 cells. The mRNA and protein levels of MASPIN were significantly higher in the MASPIN/PCR2.1 group (33.6+/-1.2, 23.4+/-1.6) than in the PCR2.1(15.0+/-1.5, 12.3+/-1.5)and the untreated group (13.7+/-2.0, 12.0+/-1.3) (P<0.05). After transfection of MASIPIN/PCR2.1, DNA apoptotic ladders appeared in SGC7901 cells and the induction of apoptosis was in a time-dependent manner. The apoptosis rates were 8.0%, 16.3% and 25.8%in the MASPIN/PCR2.1 plus TRAIL group, 3.0%, 8.2%, 14.4% in the MASPIN/PCR2.1 group, and 4.1%, 9.8%,15.9% in the TRAIL group at 12, 24, and 48 h(P<0.05). The expression levels of Bax mRNA and protein at 48 h after MASIPIN/PCR2.1 transfection were significantly higher in MASPIN/PCR2.1 plus TRAIL group(55.3+/-2.1, 75.4+/-1.3) than in the PCR2.1 group (34.3+/-1.2, 40.7+/-1.8) and the TRAIL group (43.2 +/-1.8,36.2+/-1.3)(P<0.05). The expression of Bcl-2 mRNA in the MASPIN/PCR2.1 plus TRAIL group, PCR2.1 group and TRAIL group were 28.3+/-2.5, 34.3+/-1.2, 32.8+/-2.1, respectively (P<0.05), and those of Bcl-2 protein were 17.4+/-1.5, 45.1+/-2.1, 42.8+/-1.5 in the three groups, respectively (P<0.05).


CONCLUSIONS
upregulation of MASPIN/PCR2.1 can significantly enhance the sensibility of gastric cancer cell line SGC7901 to the apoptosis inducer. This maybe related to the upregulation of Bax and downregulation of Bcl-2.
Zheng
Ai-Hua
AH
Department of Pathology, Chongqing Medical University, Chongqing, China.
Yi
Yong-Fen
YF
Zhou
Wen-Wen
WW
D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015843:Serpins / Q000235:genetics / Q000378:metabolism*; D013274:Stomach Neoplasms / Q000378:metabolism* / Q000473:pathology*; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000494:pharmacology; D014162:Transfection; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism
D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D011994:Recombinant Proteins; C085344:SERPIN-B5; D015843:Serpins; D053221:TNF-Related Apoptosis-Inducing Ligand; D051028:bcl-2-Associated X Protein