The Effect Of Rnai In Mediated PDGF-D Gene Silence On Human Gastric Carcinoma Sgc-7910 Cells

PARTⅠEXPRESSION AND SIGNIFICANCE OF PDGF-D AND VEGF IN GASTRIC CARCINOMAObjective To investigate the expression of Platelet-derived growth factor-D(PDGF-D) and Vascular endothelial growth factor(VEGF) in gastric carcinoma and elucidate the relationship between their overexpression and clinical pathological characteristics.Methods Immunohistochemical assay was performed to detect the protein expression of PDGF-D and VEGF in tissues. Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate the mRNA expression of PDGF-D and VEGF in 40 random selecting gastric carcinoma samples. The correlation between the expression of PDGF-D and VEGF, and the relationship between the expression of PDGF-D, VEGF and the clinical pathological characteristics were analyzed. Results The protein expression of PDGF-D and VEGF in gastric carcinoma tissues were significantly higher than that in adjacent tissues and normal gastric mucosa(P<0.05 );The expression of PDGF-D and VEGF correlated with TNM staging, depth of invasion,and lymphatic metastasis (P < 0.05), while the expression of PDGF-D also correlated with histological differentiation (P<0.05). There was a significant positive correlation between PDGF-D and VEGF at the mRNA and protein expression levels. (P<0.05).Conclusions PDGF-D and VEGF specifically highly expressed in gastric carcinoma. The abnormal expression may play an important role in the carcinogenesis and metastasis of gastric carcinoma.PARTⅡTHE STUDY OF RNAI INHIBITING THE EXPRESSION OF PDGF-D IN SGC-7901 CELLSObjective To construct the plasmid containing short hairpin RNA(shRNA) of PDGF-D ,to determine the inhibitory effect on the PDGF-D expression and to select stably transfected cells with G418-sulfate. Methods Two pair of reverse repeated motif of PDGF-D target sequence and one pair of control sequence were synthesized, which were inserted into pGPU6/GFP/Neo to generate the PDGF-D-shRNA plasmid, which were transfected into SGC-7901. The mRNA and protein expression of PDGF-D were examined by semiquantitative reverse transcription polymerase chain reaction(RT-PCR) and Western Blot.The plasmid with high inhibition efficiency and SGC-7901 with stable shRNA expression targeting PDGF-D was selected with G418.Result The recombinant plasmid PDGF-D-shRNA was identified by enzyme digestion and DNA sequencing.After it was transefected at 48 hour, the eukaryotic expression vectors of PDGF-D-shRNA1 and PDGF-D-shRNA2 could effectively inhibit PDGF-D expression in human gastric carcinoma cell line SGC-7901, and the latter was better(P <0.05).The level of PDGF-D mRNA and protein decreased by 68.2%, 65.2% respectively compared with that of the untreated group and the negative control group(P<0.05). Stably transfected cells were selected successfully.Conclusion The recombinant plasmid PDGF-D-shRNA2 can effectively suppress PDGF-D expression in human gastric carcinoma cell line SGC-7901. Stably transfected cells were selected successfully for next research. PARTⅢTHE EFFECT OF RNAI IN MEDIATED PDGF-D GENE SILENCE ON THE BIOLOGICAL CHARACTERISTICS OF HUMAN GASTRIC CARCINOMA SGC-7910 CELLSObjective To investigate the effect of downregulating the expression of PDGF-D on the proliferation, adhesion, migration, invasion and angiogenesis of human gastric carcinoma cell line SGC-7901 in vitro and its mechanism.Methods Cell proliferation and cell cycle were examined by MTT assay and flow cytometry, respectively. Adhesive rate and migration capability were evaluated by Matrigel adhesive assay and migration assay, respectively. Invasive capability and the effect on the tube formation of human umbilical vein endothelial cells were evaluated by invasion assay. The changes of PDGF-D signaling pathway related factor expression were measured by Western Blot and ELISA.Results After the downregulation of PDGF-D expression, proliferation, adhesion, migration, invasion of SGC-7901 and the tube formation of human umbilical vascular endothelial cells were inhibited to some degree(P<0.05);Flow cytometry analysis showed that the number of cells in S phase decreased while the number of cells in G0/G1 phase increased (P<0.05). The protein expression of Cyclin D1, MMP-2, MMP-9, VEGF andβ-catenin in nucleus decresed;The concentration of VEGF in supernate also decreased obviously(P<0.05).Conclusion The downregulation of PDGF-D expression may obviously inhibit the proliferation, adhesion, migration, invasion of SGC-7901 and the tube formation of human umbilical vascular endothelial cells in vitro.The mechanism may be that the downregulation of PDGF-D expression leads to the downregulation ofβ-catenin in nucleus,which leads to the decreased protein expression of Cyclin D1,MMP-2 ,MMP-9,VEGF.PARTⅣEFFECT OF RNAI IN MEDIATED PDGF-D GENE SILENCE ON THE GROWTH AND ANGIOGENESIS OF HUMAN GASTRIC CARCINOMA XENOGRAFT IN NUDE MICEObjective To investigate the effect of RNA interference in mediated PDGF-D gene silence on the growth and angiogenesis of human gastric carcinoma Xenograft in nude mice.Methods The effect of PDGF-D gene silence on the growth and the volumes of human gastric carcinoma Xenograft in nude mice were observed. The expression of PDGF-D and VEGF protein in tumor tissues was measured by Western Blot and microvessel density (MVD) were detected by immunohistochemistry.Results The tumor grew slowly after PDGF-D gene silence. The volumes of human gastric carcinoma Xenograft in nude mice was much smaller than that in the control groups.The expression of VEGF downregulated and MVD was reduced.Conclusion PDGF-D gene silence may inhibit the growth of human gastric carcinoma Xenograft in nude mice, which might be related with dowm-regulation of VEGF to inhibit the angiogenesis of Xenograft.