Construction Of The Recombinant Prokaryotic Expression Plasmid PET-21a (+) -DCN And The Influences On BGC-823 Gastric Cancer Cell Line

Background Decorin is a leucine-rich and ubiquitous proteoglycan that is a normal component of the extracellular matrix and has recently been described as capable of retarding the growth of various tumor cell line, both in vitro and in vivo.The cDNA of decorin extron, was composed of 1080 bp of nucleotides and encoded a protein of 395 amino acids. Objects Our aim is to clone and expresse the human decorin extron gene, to evaluate the anti-proliferation on BGC-823 gastric cancer cell line and influence on mutational P53 and VEGF expression using decorin. Methods Firstly, using decorin specific primer and by RT-PCR, the full length of cDNA of decorin extron was obtained through human muscle, then recombined plasmid -decorin was constructed and sequenced to identify the gene sequence. Furthermore, pET-21a(+)-decorin was transformed into E.coli BL21(DE3)and after higher temperature and IPTG induction, the fusion protein was expressed. After being separated by SDS-PAGE electrophoresis, being eluted and purified through dialysis, the purification of fusion protein pET-21a(+)-decorin was obtained. Secondly, After a period when BGC-823 gastric cancer cell line were treated with decorin in different concentrations, the suppressive effect of decorin on BGC-823 gastric cancer cell line were detected by the MTT assay and counting cell. The morphological changes of gastric cancer cell were observed by inverted microscope and HE stain. The positive expression of mutational P53 and VEGF was determined by immunocytochemistry(ICC). Results The recombinant vector was constructed and identified using genetic cloing technology, and the target peptide was produced in E.coli BL21(DE3)in this experiment. The fusion protein was high expressed. After a period when BGC-823 gastric cancer cell line were treated with decorin in different concentrations, the proliferation of BGC-823 gastric cancer cell line were inhibited by decorin respectively showed by MTT assay and counting cell. These results demonstrated that had a dosage and time dependent relationship. There was a statistically significant decreased in the proliferation rate among the three groups with different concentrations(P<0.05 或P<0.01). The morphological changes of gastric cancer cell that the microvillus disappeared, cytoplasm had vacuolation, chromation were pyknosis and cogulate to mass clustered around the membrane were revealed by invert microscope and HE stain.The expressions of mutational P53 and VEGF were decreased and had a concentration and time dependenent relationship by immunocyto-chemistry(ICC). The positive expression rate was differs significantly(P<0.05 或P<0.01)among the three groups with different concentration. Conclusion The prokaryotic expression vector with pET-21a(+)-decorin can effectively expression decorin fusion protein. The experiment will establish the basis for studying the mechanism of the anti-tumor function of decorin. Our study indicated that decorin can induce the anti-proliferation of BGC-823 gastric cancer cell line and the molecular biological mechanism may be related to the suppressing the expression of mutational P53 and VEGF.