The Function And Mechanism Investigations Of TIPE1 Inhibiting Angiogenesis In Gastric Cancer

Objects: TIPE1(TNFAIP8L1,TNF-?-induced protein 8-like 1),a member of the TNFAIP8 family,functions as a tumor suppressor gene in a variety of tumors through inhibiting tumor cell proliferation and promoting tumor cell apoptosis,but the function and mechanism of TIPE1 in regulating tumor angiogenesis is unknown.In this thesis,we mainly investigate the role and molecular mechanism of TIPE1 in regulating gastric cancer angiogenesis.Methods: First,multiple gastric cancer cells and normal gastric epithelial cells were cultured and total m RNA and protein were extracted for determining the expression of TIPE1 in each of the above cells by quantitative PCR and Western blotting.The plasmid-based TIPE1 overexpression system was used to transfect the TIPE1-lowexpression gastric cancer cells and the TIPE1 stably overexpressed MGC-803 cells were selected by puromycin(named MGC-803-TIPE1 cells).MGC-803-Ctrl and MGC-803-TIPE1 cells will be subcutaneously injected into Balb / c nude mice to establish a subcutaneous xenograft model and the anti-tumor acitivaty of TIPE1 on gastric cancer was analyzed.The tumor tissues were embedded in paraffin and sliced and used for immunofluorescence staining(CD31 staining).Furthermore,HUVEC-related cell viability assay,tube formation assay and invasion assay were performed to investigate the anti-angiogenesis ability of TIPE1 in gastric cancer.To investigate the molecular mechanism under TIPE1 inhibiting tumor angiogenesis,Western blotting,q PCR and ELISA assay were performed to detect the VEGF-A m RNA,intracellular and extracellular VEGF-A protein in MGC-803-Ctrl,MGC-803-TIPE1,SGC-7901-Ctrl and SGC-7901-TIPE1 cells.Furthermore,HUVEC proliferation assay,Matrigel-mediated HUVEC tube formation assay and Matrigel-mediated HUVEC invasion assay were performed to determine the necessary role of VEGF-A during TIPE1 inhibiting angiogenesis.Results: The results of Western blotting and quantitative PCR showed that TIPE1 was low or no expression in the gastric cancer cells.The results of subcutaneous xenograft in MGC-803 showed that overexpression of TIPE1 could significantly inhibit the growth of MGC-803 tumor by 63.5% in tumor volume(tumor volume: MGC-803-Ctrl 895.4 ± 85.6 mm3 vs MGC-803-TIPE1 326.8 ± 42.7 mm3);by 67.9% in tumor weight(tumor volume: MGC-803-Ctrl 0.81 ± 0.13 g vs.MGC-803-TIPE1 0.26 ± 0.09 g).CD31 immunofluorescence staining and HUVEC-related assay confirmed the inhibition role of TIPE1 in gastric cancer angiogenesis.ELISA,q PCR and Western blotting results showed that TIPE1 could inhibit the expression of VEGF-A m RNA and protein,but has no significant effect on the expression of b FGF,VEGF-C,HGF,VEGF-D and PDGF-BB.Restoring the VEGF-A expression in MGC-803-TIPE1 cell culture supernatant would efficiently blocked the inhibitory effect of TIPE1 on HUVEC proliferation,tubular analog formation and cell invasion.Conclusions: TIPE1 is in low or no expression in MGC-803,SGC-7901,BGC-823,MKN45,AGS gastric cancer cells.Overexpression of TIPE1 in gastric cancer cells significantly inhibited the growth of MGC-803 subcutaneously transplanted tumors and tumor angiogenesis in the subcutaneously transplanted tumor tissues;the supernatant of MGC-803-TIPE1 cells significantly inhibited the proliferation of HUVEC cells,the formation of tubular analogs,and cell invasion.Overexpression of TIPE1 significantly inhibited the expression of VEGF-A in gastric cancer.Further studies showed that VEGF-A is the key molecular node of TIPE1 in inhibiting angiogenesis of gastric cancer.The experimental results of this paper will provide an experimental basis and theoretical basis for further understanding the role of TIPE1 in the development and progression of gastric cancer,and provide new targets and strategies for the diagnosis and treatment of gastric cancer.