[Interference of human Na/K-ATPase B1 subunit on proliferation and migration of gastric adenocarcinoma cell line SGC-7901].
OBJECTIVE
The Na+/K+ ATPase B1 (ATP1B1) subunit gene is highly expressed in well-differentiated tumor cells, while it is hypoexpressed in poorly differentiated tumor cells. The expression of ATP1B1 is closely related to cell tight junction and polarity of epithelial cells. This study was to investigate the effect of specific interference of human Na+/K+ ATP1B1 using shRNA on cell proliferation and migration of gastric adenocarcinoma cell line SGC-7901.


METHODS
Four shRNA plasmids specifically targeting different fragments of ATP1B1, sh150, sh295, sh562, sh765, were constructed and transiently transfected into SGC-7901 cells. Stable positive clones, shATP1B1-7901 cells, were sorted out by G418. The expression of ATP1B1 mRNA was detected by semi-quantitative RT-PCR and real-time PCR. Cell proliferation was measured by MTT, cell cycle distribution was assessed by flow cytometry, and clone forming was analyzed by the colony formation assay. Cellular migration was observed using the Transwell experiment.


RESULTS
At 24 h after transfection, the inhibition ratios of sh150, sh295, sh562, sh765 on ATP1B1 mRNA were (60.87+/-4.38)%, (44.93+/-2.24)%, (52.17+/-2.60)% and (52.17+/-2.60)% respectively in SGC-7901 cells, which were significantly higher than that of shNC control (3.00+/-0.15)%(p<0.05). Among the four ATP1B1 shRNAs, sh150 exerted the strongest effect (p<0.05) and was used in the following study. Assessed by RT-PCR and real-time PCR, the expression of ATP1B1 mRNA was inhibited by (85.72+/-5.22)% and (87.53+/-3.23)% in the shATP1B1-7901 group, in comparison with (3.3+/-0.22)% and (4.17+/-0.33)% in the shNC-7901 group (p<0.05). The proliferation ratio was higher in the shATP1B1-7901 group than in the shNC-7901 and SGC-7901 groups 3 days after transfection (p<0.05). Cells at S and G2/M phases in the shATP1B1-7901 group were significantly increased compared with those in the shNC-7901 and SGC-7901 groups (p<0.05). The clone formation rate of the shATP1B1-7901 group (68.50+/-2.65)% was higher than that of the shNC-7901 group (50.00+/-2.53)% and of the SGC-7901 group (52.50+/-2.11)% (p<0.05). Moreover, the migration ratio of the shATP1B1-7901 group(2.80+/-0.02)% was significantly enhanced comparing to the shNC-7901 (1.15+/-0.05)% and the shNC-7901 groups (1.25+/-0.02)% (p<0.05).


CONCLUSIONS
Silencing of ATP1B1 gene can enhance the proliferation and migration capability of SGC-7901 cells.
Liu
Kai
K
Department of Geraeology, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, Sichuan, P. R. China.
Zhang
Jie
J
Ren
Jing-Jing
JJ
Wang
Xiu-Jie
XJ
Yang
Hong-Liang
HL
Lin
Ping
P
D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002453:Cell Cycle; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D006801:Humans; D010957:Plasmids; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics* / Q000378:metabolism; D013274:Stomach Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D014162:Transfection
C503551:ATP1B1 protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000254:Sodium-Potassium-Exchanging ATPase