[Construction of a gastric cancer related gene GCRG213-specific siRNA expression vector and its effect on gastric cancer cells in vitro].
OBJECTIVE
To investigate the effect of gene GCRG213 siRNA transfection into gastric cancer cell line MKN45 cells.


METHODS
Two pairs of DNA sequences containing small hairpin structure to GCRG213 were designed and synthesized. The complement form was obtained by annealing and inserted into RNAi expression vector IMG-800. They are IMG-800-1 and IMG-800-2 correspondingly. The recombinant plasmid IMG-800-1, IMG-800-2 and the vector IMG-800 were separately transfected into MKN45 cells conducted by lipofectamine 2000. After G418 selecting, the cells were transfected steadily. Expression of GCRG213 was detected by semi-quantitative RT-PCR and Western Blot. The growth graph of six steady transfected cell cultures was protracted by cell counting. FACS was used to detect the cell cycle, and Annexin V FITC/PI double labeling were used to detect the effects on cell apoptosis in the above-mentioned cells. The clone formation rate in plate and in nude mice was tested to investigate the tumorigenic characteristics of the six steadily transfected cells in vitro and vivo.


RESULTS
Through sequencing, two pairs of DNA sequences containing small hairpin structure to GCRG213 were proved to be successfully cloned into siRNA expression vector IMG-800, correspondingly called IMG-800-1 and IMG-800-2. The recombinant plasmid IMG-800-1, IMG-800-2 and vector IMG-800 were transfected separately into MKN45 cells conducted by lipofectamine 2000. After G418 selecting, the cells were transfected steadily. Transfecting the siRNA vector (IMG-800-1, IMG-800-2 ) into the MKN45 cells significantly decreased the expression of GCRG213, at both mRNA and protein levels. The growth graph showed that the growth of IMG-800-1 and IMG-800-2 transfected cells were slower than that of vector transfected cells. The proportion of cells in G2/M and/or S phase decreased in the cells transfected with IMG-800-1 and IMG-800-2 and cell apoptosis increased. The average clone formation rate in vitro decreased in the cells transfected with IMG-800-1 and IMG-800-2, compared with those transfected with vector. In vivo, the time of tumor formation of IMG-800-1 and IMG - 800-2 transducted cells in nude mice was prolonged and the tumor size was smaller.


CONCLUSIONS
GCRG213 SiRNA transfection may induce inhibition of growth and proliferation of tumor cells, promote cell apoptosis, and inhibit the tumorigenicity in vitro and vivo.
Gao
Li-Li
LL
Department of Gastroenterology, General Hospital of PLA, Beijing 100853, China. GLL301818@yahoo.com.cn
Wu
Ben-Yan
BY
Wang
Meng-Wei
MW
Zhan
Da-Wei
DW
Zhang
Lin
L
You
Wei-Di
WD
Wang
Wei-Hua
WH
D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D005260:Female; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D036361:Peptide Hormones / Q000096:biosynthesis* / Q000235:genetics; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D014162:Transfection; D014183:Transplantation, Heterologous
C477000:GKN1 protein, human; D036361:Peptide Hormones; D012333:RNA, Messenger; D034741:RNA, Small Interfering