The Effect Of Blocking VEGF-C Combined With Bevacizumab On Malignant Biological Behavior Of Gastric Cancer

Background and Objective Gastric cancer(GC)is one of the malignant tumors that threaten to human life and health.However,its pathogenesis is still not fully clarified and its prevention and treament are still unsatisfactory.Surgery is still the only way to cure resectable gastric cancer currently.Modern cancer molecular biology studies have proved that the occurrence and progression of cancers are closely related to gene mutation.Therefore,targeted therapy for cancer driven genes has great potential which likely to cure cancers.But target therapy of gastric cancer is relatively lagging.Though To GA studies,which reported by American Society of Clinical Oncology(ASCO),2009,had confirmed that trastuzumab combined with chemotherapy can improve the survival of Her2 positive patients with advanced gastric cancer,the beneficiaries were in the minority.Therefore,targeted therapy of gastric cancer deserves further research.Bevacizumab(Bev)is a human derived vascular growth factor which have a powerful antiangiogenic effect.Bevacizumab plays a good antitumor role in colorectal cancer,lung cancer and renal cell carcinoma but gastric cancer.Some scholars believe that gastric cancer relies deeply on dual lymphatic and vascular pathways for its proliferation,invasion and metastasis.(the proliferation and invasion of gastric cancer are mainly dependent on the dual pathways of lymphatic vessels and vessels.)For this reason,the combined intervention by blocking lymphatic vessels and angiogenesis simultaneously is more effect than a single intervention on controlling tumor proliferation,invasion and metastasis,and has a potential application prospect.Therefore,our study investigated the effects of blocking the expression of VEGF-C by utilizing RNAi technique combined with bevacizumab(VEGF-A-targeted inhibitor)on malignant biological behaviors of gastric cancer to design a novel and effective strategy for the treatment of GC.Methods Part 1 Real-time quantitative PCR(RT-q PCR)was used to detect the m RNA expression of VEGF-A and VEGF-C in three gastric cancer cell lines(SGC7901,MGC803 and BGC823)and a normal gastric epithelial cell line(GES-1).Gastric cancer cell lines with relatively high expression of VEGF-A and VEGF-C m RNA was selected for further experiments.Part 2 CCK-8 proliferation toxicity test was applied to examine growth inhibition rates and IC50 values of bevacizumab on gastric cancer cell lines.VEGF-C m RNA sequences were searched in NCBI,then we designed and compounded 3 groups of VEGF-C-sh RNA primers.The empty lentivirus and lentivirus carrying VEGF-C-sh RNA were transfected into gastric cancer cell lines,respectively.Western Blot was utilized to select VEGF-Cshi RNA sequence which has the highest inhibition efficiency for VEGF-C.Gastric cancer cells stably transfected with this VEGF-C-sh RNA was select for subsequent experiments.Part 3 Cells were divided into five experimental groups: Control group(nontransfected,no bevacizumab);Empty vector group(transfected with empty vector,no bevacizumab);VEGF-C-shi RNA group(transfected with VEGF-C-shi RNA,no bevacizumab);Bevacizumab group(nontransfected,bevacizumab);Combination group(transfected with VEGF-C-shi RNA,bevacizumab).Cell proliferation(CCK-8 assay,colony forming experiment),apoptosis(flow cytometry),cell invasion(transwell assay)and migration(scratch assay)were evaluated.The m RNA and protein levels of VEGF-A adn VEGF-C were confirmed via RT-q PCR and Western blotting,respectively.Rusults Part 1 The relative expression of VEGF-A m RNA in three GC cell lines(SGC7901,MGC803 and BGC823)and the normal gastric epithelial cells(GES-1)were 3.847�0.208,6.667�0.74,5.599�1.4 and 1.059�0.114,respectively.The relative expression of VEGFC m RNA in cell lines mentioned above were 3.698�0.16,9.005�0.397,36.779�0.664 and 1.016�0.011,respectively.The expression levers of VEGF-A and VEGF-C m RNA in GC cell lines(SGC7901,MGC803 and BGC823)were significantly higher than that in normal gastric epithelial cells GES-1(P <0.05).The GC cell line SGC7901,which has a relatively high expression lever of VEGF-A and VEGF-C m RNA,was used as a target gastric cancer cell line for subsequent experiments.Part 2 CCK-8 proliferation toxicity test showed that the inhibitory effect of bevacizumab on the target gastric cancer cell line SGC7901 was proportional to the drug concentration.After adding 0 ug/ml,500 ug/ml,1000 ug/ml,1500 ug/ml,2000 ug/ml and 2500 ug/ml bevacizumab into gastric cells for 48 h,the growth inhibition rates were 0,0.438�0.013,0.678�0.004,0.729�0.004,0.75�0.003 and 0.818�0.002,respectively.The growth inhibition rates of cells treated with different concentrations of bevacizumab were significantly higher compared with those in group without bevacizumab.Inhibition rate diagram was drew.The IC50 value was 160ug/ml for 48 h.The VEGF-C protein expression in gastric cancer cells transfected with no-load lentivirus,VEGF-C-sh RNA1,VEGF-C-sh RNA2 and VEGF-C-sh RNA3 were 1.002�0.003,0.0783�0.007,0.885�0.065 and 0.423�0.014,respectively.For the sh RNA3 had the highest blocking efficiency of VEGF-C protein expression,we selected gastric cancer cell line SGC7901 transfected with VEGF-C-sh RNA 3-load vector for subsequent experiments.Part 3 CCK-8 proliferation toxicity test showed that the inhibitory effect of bevacizumab on the target gastric cancer cell line SGC7901 was proportional to the drug concentration.After adding 0 ug/ml,500 ug/ml,1000 ug/ml,1500 ug/ml,2000 ug/ml and 2500 ug/ml bevacizumab into gastric cells for 48 h,the growth inhibition rates were 0,0.438�0.013,0.678�0.004,0.729�0.004,0.75�0.003 and 0.818�0.002,respectively.The growth inhibition rates of cells treated with different concentrations of bevacizumab were significantly higher compared with those in group without bevacizumab.Inhibition rate diagram was drew.The IC50 value was 160ug/ml for 48 h.The VEGF-C protein expression in gastric cancer cells transfected with no-load lentivirus,VEGF-C-sh RNA1,VEGF-C-sh RNA2 and VEGF-C-sh RNA3 were 1.002�0.003,0.0783�0.007,0.885�0.065 and 0.423�0.014,respectively.For the sh RNA3 had the highest blocking efficiency of VEGF-C protein expression,we selected gastric cancer cell line SGC7901 transfected with VEGF-C-sh RNA 3-load vector for subsequent experiments.Conclusion The expression of VEGF-A and VEGF-C in gastric cancer cells was significantly increased.RNAi can effectively block the expression of VEGF-C in gastric cancer cell line SGC7901.Bevacizumab can inhibit the activity of gastric cancer cell line SGC7901.RNAi block VEGF-C expression combined with bevacizumab may simultaneously inhibit lymphangio genesis and angiogenesis,thereby more effectively inhibiting the proliferation,invasion and migration of gastric cancer cell line SGC7901 and potently inducing apoptosis of tumor cells.This topic provides a new target and treatment strategy for the basic research of gastric cancer and the development of new clinical drugs.