Study Of Expression Of Vascular Endothelial Growth Factor C Mrna In Non-Small Cell Lung Cancer And The Action Of Its Antisenseoligodeoxyribonucleotid On Lung Carcinoma Cell

Lung cancer is one of the most common malignant tumor whieh seriously Threaten human life and health intoday's world. Lung cancer has beeome the Leading cause of death.Of about 80% patients is non-small-cell lung caneer(NSCLC) of which diagnosed at advaneedstage, unresectable about 80%.The treatment of NSCLC include surgery,chemotherapy,radiotherapy and Molecular targeted therapy, whatever the treatmen trequires a effective index of guide and judge for the treatment of NSCLC.TNM staging are widely used as the current indieators to evaluate the treatlnent and prognosis of NSCLC, which provide some guidance value.However,in clinical work,a part of NSCLC patients who although have been the early diagnosis and treatment appeared in the short term recurrence and metastasis, The biologieal behavior of tumor charaeteristies of these patients are no doubt more aggressive side.Therefore,it is obviously not enough only depending onTNM staging of lung cancer.Studies have found that tumor growth, invasion and metastasis are dependent on angiogenesis processes, which are closely related to the prognosis of cancer.Thesame as other solid tumors, turmor angiogenesis provide nutrients to promote tumor growth, tumor invasion and metastasis. Tumor angiogenesis involves a variety of different gene mutations and expression of cytokines and their receptors and is regulated both positive and negative factors. In tumor tissues, as pro-angiogenie factors are dominant, Vaseular endothelial gowth faetor (VEGF) is widely recognized as the most important angiogenic factors, and plays an important role in the promotion of tumor microvessels and Lymhangiogenesis. Therefore, the understanding of lung VEGF expression and angiogenesis may help clarify the mechanism of growth and metastasis of lung cancer. VEGF-C antisense oligodeoxyribonucleotid may inhibit endothelial cell proliferation and inhibit angiogenesis, prevent cell adhesion, proliferation and migration, and blocking the violations of tumor from vessels and tissues.Objective:To study the expression of vascular endothelial growth factor C(VEGF-C) mRNA in non-small cell lung cancer(NSCLC). VEGF-C antisense oligodeoxyribonucleotid (ASODN) mediated by bangosome was transfected into human lung cancer cell strain A-549.To examine the invasive ability in vitro and expression of VEGF-C protein.Methods:(1) Complementary deoxyribonucleic acid was prepared by gene c loning technique,directed cloning and put into plasmid,and was transformed into competent Bacillus coli to get positive converter of recombinant plasmid.Probe o f VEGF-C complementary RNA was made and labeled by digolan in vitro.Expre ssion of VEGF-C mRNA was examined in NSCLC by hybridization technique. (2)VEGF-C ASODN was transfectde into human lung cancer cell line A-549, th e ability of its adhere and invasion was detected,and expression of VEGF-C prot ein was examined by Western blot technique.Results:(1)Total RNA,which can synthesize a cDNA chain and with VEGF-C gene-specific primers,could be used as a template for PCR amplification, the size of a specific band of about 1.1 kb emerged. A small amout of the plasmid preparation were digested by restriction enzyme EcoR I and Xho I, the inserted fragment of about 1.1kb could be observed in recombinant plasmid.In the positive clone, the inserted fragemts were all sequenced,and sequences were as same as the human VEGF-C gene cDNA.VEGF-C mRNA expressed in lung squamous cell carcinoma and adencarcinoma in some degree,and it mainly distributed in lung cancer cells,vascular endothelial cells and fibroblast.Positive expression rate were 65.4%(17/26) and 59.1% (13/22), respectively. (2)Adhere rate of lung cancer cells transfected by VEGF-C ASODN degraded obviously (0.15±0.01,0.29±0.02,0.49±0.02,0.76±0.01),it is different significantly compared to control group(P<0.01).Cell population of infiltration in 48 hours reduced obviously (58.6±9.2),and invasive ability to recombinate basal membrane attenuated obviously(P<0.01).The expression of VEGF-C protein descend obviously(P＜0.01).Conclusions:VEGF-C mRNA can expression in NSCLC in some degree; (2)VEGF-C ASODN can inhibit invasion ability of lung cancer cells in vitro by down regulation of expression of VEGF-C protein. PATR1CLONING OF VASCULAR ENDOTHELIAL GROWTH FACTOR C GENE AND EXPRESSION OF ITS MRNAIN NON-SMALL CELL LUNG CANCER TISSUEObjective: To study the expression of vascular endothelial growth factor C(VEGF-C) mRNA in non-small cell lung cancer(NSCLC).Methods:(l) Complementary deoxyribonucleic acid was prepared by gene c loning technique.(2)The cDNA was directed cloning and put into plasmid,and wa s transformed into competent Bacillus coli to get positive converter of recombina nt plasmid.Probe of VEGF-C complementary RNA was made and labeled by dig olan in vitro.(3) Expression of VEGF-C mRNA was examined in NSCLC by hy bridization technique.Results:(l)Total RNA,which can synthesize a cDNA chain and with VEGF-C gene-specific primers,could be used as a template for PCR amplification, the size of a specific band of about 1. lkb emerged.(2)A small amout of the plasmid preparation were digested by restriction enzyme EcoR I and Xho I, the inserted fragment of about 1.1kb could be observed in recombinant plasmid.(3)In the positive clone, the inserted fragemts were all sequenced,and sequences were as same as the human VEGF-C gene cDNA.(4)VEGF-C mRNA expressed in lung squamous cell carcinoma and adencarcinoma in some degree,and it mainly distributed in lung cancer cells,vascular endothelial cells and fibroblast.Positive expression rate were 65.4% (17/26) and 59.1% (13/22),respectively.Conclusions: VEGF-C mRNA can expression in NSCLC in some degree; PART 2STUDY OF INVASIVE INHIBITION OF VASCULARENDOTHELIAL GROWTH FACTOR C ANTISENSEOLIGODEOXYRIBONUCLEOTID ON HUMAN LUNGCANCER CELL LINEObjective: VEGF-C antisense oligodeoxyribonucleotid (ASODN) mediated by bangosome was transfected into human lung cancer cell strain A-549.To examine the invasive ability in vitro and expression of VEGF-C protein.Methods: (1)VEGF-C ASODN was transfectde into human lung cancer eel 1 line A-549, the ability .of its adhere and invasion was detected.(2) Expression of VEGF-C protein was examined by Western blot technique.Results: (1) Adhere rate of lung cancer cells transfected by VEGF-C ASODN degraded obviously (0.15±0.01, 0.29±0.02, 0.49±0.02, 0.76±0.01) ,it is different significantly compared to control group(P<0.01).(2) Cell population of infiltration in 48 hours reduced obviously (58.6±9.2),and invasive ability to recombinate basal membrane attenuated obviously(P<0.01).(3)The expression of VEGF-C protein descend obviously (P<0.01).Conclusions: VEGF-C ASODN can inhibit invasion ability of lung cancer cells in vitro by down regulation of expression of VEGF-C protein.