[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.

CONCLUSION: GCRG213 SiRNA transfection may induce inhibition of growth and proliferation of tumor cells, promote cell apoptosis, and inhibit the tumorigenicity in vitro and vivo.