[Construction of recombinant adenovirus vector expressing shRNAs targeting COX-2, AKT1 and PIK3R1 gene and its inhibition effect on proliferation of human gastric adenocarcinoma].
OBJECTIVE
To construct a recombinant adenovirus vector that expresses small hairpin RNAs (shRNA) against COX-2, AKT1 and PIK3R1 gene and to evaluate its potential for suppressing the cell proliferation of human gastric adenocarcinoma SGC701 cell in vitro and in vivo, which will enable the development of a gene therapy protocol for the treatment of human gastric adenocarcinoma.


METHODS
Three strips of shRNA targeting AKT1, COX-2 and PIK3R1, was subcloned into adenovirus expression vector. After verification, it was amplified and titered. The recombinant adenovirus expression vector was infected into human gastric adenocarcinoma SGC7901 cells in vitro and the infected cells were injected in nude mice. The mRNA and protein expression levels of AKT1, COX-2 and PIK3R1 were determined by real-time PCR and Western blot respectively. Cell proliferation in vitro was determined by methyl thiazolyltetrazolium (MTT) assay and flow cytometry, tumor growth in vivo was measured by volume of tumor in nude mice.


RESULTS
Restriction digestion and sequencing analysis showed that the rAd5-C-A-P adenovirus expression vector was constructed successfully. It significantly inhibited the expression of AKT1, COX-2 and PIK3R1, and cell growth was inhibited over 70% as indicated by MTT assay and accompanied with G0/G1 phase arrest. Cell growth on matrigel matrix showed that the rAd5-C-A-P transfected cells were detached from the matrix or grew in a scattered clustering pattern, indicating poor cell growth activities in 2-D matrigel. Tumor growth in nude mice in the C + A + P group was inhibited (P<0.01).


CONCLUSIONS
shRNA targeting COX-2, AKT1 and PIK3R1 down regulated significantly the expression of the three genes in a sequence-specific manner, exerted proliferation inhibition effect on SGC7901 cells in vitro and in vivo.
Fu
Yan-chao
YC
Department of Gastroenterology, Tianjin Medical University General Hospital, Tianjin, 300052 P.R. China.
Zhang
Jing
J
Zhang
Kai-ru
KR
Zhang
Qing-yu
QY
Kang
Chun-sheng
CS
Wang
Tao
T
D000230:Adenocarcinoma / Q000235:genetics* / Q000503:physiopathology / Q000628:therapy; D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D051546:Cyclooxygenase 2 / Q000235:genetics* / Q000378:metabolism; D004195:Disease Models, Animal; D015536:Down-Regulation; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D055029:Inverted Repeat Sequences; D051379:Mice; D008819:Mice, Nude; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics*; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000627:therapeutic use*; D013274:Stomach Neoplasms / Q000235:genetics* / Q000503:physiopathology / Q000628:therapy
D034741:RNA, Small Interfering; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D019869:Phosphatidylinositol 3-Kinases; C494918:AKT1 protein, human; D051057:Proto-Oncogene Proteins c-akt