Role Of Angiopoietin-1 In Tumorigenesis And Angiogenesis Of Gastric Cancer

The current studies were planed to clone the cDNA sequence of human angiopoietin-1 (Angl) encoding gene by RT-PCR method and to construct its sense, antisense eukaryotic expression vectors by directional cloning. Three modified SGC7901 cell lines were established by stable transfection with recombinant vectors or empty vector as control using lipofectin method after screenig for two months. The efficiency of transfection was verified by detecting Angl expression level in modified SGC7901 cells with both semiquantitative PCR in mRNA level and Western Blotting in protein level. The effect of Angl on HUVEC was measured by MTT assay and flow cytometry and its roles in tumorigenesis and angiogenesis of gastric cancer were evaluated by in vivo nude mice tumor transplantation test measuring weight and volume of tumor graft and microvasculature density. The main methods and results were the following:1. Human Angl cDNA was obtained by RT-PCR method from human placental tissues. The PCR product was verified by DNA sequencing.2. The recombinant sense and antisense eukaryotic expression vectors pcDNA3.1 - Angl+ and pcDNA3.1 - Angl- were constructed by directional cloning the target gene into eukaryotic expression vector pcDNAS.l-Vj-HisC. The direction was confirmed by endonuclease digestion.3. pcDNA3.1-Angl+ was transiently transfected in 293 cells and the supernatant was harvested after 48h, then added to the culture media of HUVEC. The effect of Angl on proliferation and apoptosis of HUVEC was detected by MTT colorimetry assay and PI/AnnexinV double staining, respectively. Results showed that the supernatant of transiently transfected293 cells could promote the proliferation and reduce the apoptosis of HUVEC.4. Stable transfectants were established by stable transfection, namely 7Angl+ for sense, 7Angl- for antisense and 790IP for empty vector using liposome mediated method after G418 selection for two months. Semiquantitative PCR and Western Blotting results testified that the mRNA and protein level of Angl were both significantly downregulated in 7Angl- cells.5. MTT assay revealed that proliferation of transfected cells wasn't altered significantly.No significant changes of cell cycle were found in all transfectants.6. Tumorigenesis assay showed that 7Angl- group grew slower than other groups and the mean tumor graft weight (mg) 30 days after implantation in nude mice were(x±SD): 293.00 ± 95.54(7Angl-), 652.67±132.07(7Angl +) and 624.00± 77.78(7901P).Statistics results showed a significant growth suppression of antisense vector transfected cells (p<0.01).7. The tumor graft of 7Angl- cell in nude mice had less(p<0.01) vessel formation with MVD (.T+SD) 6.00±1.73 compared to 7901P group MVD 8.44±1.33 and 7Angl+ group MVD 8.78±1.92.All these results indicated that the expression of Angl in human gastric cancer cell line SGC-7901 may promote the tumorigenesis process by enhancing angiogenesis. Inhibition of Angl with antisense RNA technique could inhibit the proliferation of tumor cells in vivo by reducing angiogenesis. The Angl targeted strategy may have a potential of antiangiogenesis therapy for gastric cancer.