[Inhibitory effect of adenovirus-mediated short hairpin RNA targeting P85 and Akt1 on growth of human gastric adenocarcinoma cell].
OBJECTIVE
To construct a short hairpin RNA(shRNA) adenovirus vector targeting P85 and protein kinase B1 (PKB1/Akt1) and study its effects on the growth of SGC-7901 human gastric adenocarcinoma cells.


METHODS
P85 and Akt1 shRNA expression frames were subcloned to pGSadeno adenovirus vector with homologous recombination technology to construct pGSadeno-P85 + Akt1 (rAd5-P + A) vector. After screening and amplification, the recombinant adenovirus vector was digested with PacI and transfected into SGC-7901 cells and then its titer and transfection efficiency were detected with fluorescent microscope. P85 and Akt1 mRNA protein expression was identified with real-time PCR and Western blot. The proliferative activity of tumor cells was evaluated with MTT assay and flow cytometry in vitro. rAd5-HK and rAd5-P + A mediated by adenovirus were injected into the established subcutaneous SGC-7901 gastric adenocarcinoma in nude mice. During the observation period of 21 days, tumor volume was measured every 3 days to further testify the anti-tumor effect of rAd5-P + A on the SGC-7901 gastric adenocarcinoma cells and cell in situ apoptosis was detected with TUNEL assay.


RESULTS
The adenovirus vector rAd5-P + A was successfully constructed and it dramatically downregulated P85 and Akt1 mRNA expression in SGC-7901 gastric adenocarcinoma cells. Compared with a control group of SGC-7901 cells and cells transfected with general adenovirus rAd5-HK as control, P85 and Akt1 protein expression 48 h and 72 h after rAd5-P + A transfection was decreased by 57.5% and 63.7%, 67.8% and 75.6% with statistical significance (P = 0.005, P = 0.003). Cell proliferative activity in rAd5-P + A transfected cells was suppressed from the second day (P < 0.001) and the decreased P85 and Akt1 expression was accompanied by 5.9% -7.1% decrease of S phase fraction and 12.1% - 13.7% increase of G0/G1 phase. The tumor volume of rAd5-P + A treated group was smaller than that of the control and rAd5-HK group with statistical significance (F = 9.871, P = 0.025). Moreover, rAd5-P + A could induce cell in situ apoptosis.


CONCLUSIONS
Adenovirus-mediated targeting P85 and Akt1 shRNA can inhibit the growth of SGC-7901 human gastric adenocarcinoma cells and this may provide a new strategy of combination gene therapy in gastric adenocarcinoma.
Zhang
Jing
J
Department of Gastroenterology, Tianjin Medical University General Hospital, Tianjin 300052, China.
Fu
Yan-Chao
YC
Kang
Chun-Sheng
CS
Zhang
Qing-Yu
QY
Wang
Tao
T
Zhang
Jie
J
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D045744:Cell Line, Tumor; D016503:Drug Delivery Systems; D005260:Female; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D011494:Protein Kinases / Q000235:genetics; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D013274:Stomach Neoplasms / Q000235:genetics* / Q000473:pathology*; D014162:Transfection
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011494:Protein Kinases; C494918:AKT1 protein, human; D051057:Proto-Oncogene Proteins c-akt