Effect Of NGF,VEGF And Its Receptor With Perineural Invasion And Hematogenous Metastasis In Salivary Adenoid Cystic Carcinoma

Objective:(1) Construct short hairpin RNA(shRNA) eukaryotic expression vectors targeting to nerve growth factor(NGF) and vascular endothelial growth factor(VEGF) gene and prepare for exploring the function of NGF and VEGF gene with RNA interference(RNAi) technique in salivary adenoid cystic carcinoma(SACC) tissues.(2) To analyze the expressions of tyrosine kinase-A(TrkA) and Vascular Epidemal growth factor receptor-2 (VEGFR2) in 47cases of salivary adenoid cystic carcinoma.Method:(1) constructed the plasmid that contained hU6 promoter (pGenesil-1.1).The vectors were linearized with Eco31I and the plasmids were digested with SacI and Sequencing analysised.(2)Recombinant pGenesil-1.1-NGF,VEGF vectors were transformed into competent DH5 a. After transfecting the plasmids into ACC cells with Lipofectamine 2000,The mRNA and protein expression of NGF,VEGF at the time of 48h and 72h was detected by fluorescence RT-PCR and west bloting.(3)The expressions of TrkA,VEGFR2 were detected by immunohistochemistry staining in 47 cases of Acc.Clinical data were reviewed by multivariate prognostic analysis. Result:(1) The recombinant pGenesil1.2-NGF and pGenesil1.1-VEGF plasmids were transformed into DH5αhost bacterium successfully.Sequencing analysis showed the inserted sequences were exactly correct.(2) After transfecting the plasmids into ACC cells with Lipofectamine 2000.The mRNA and protein expression of NGF and VEGF in ACC cells significantly decreased with the RNA interfered which compared with the negative control.(3)The positive rate of TrkA and VEGFR2 in ACC was 87.23%(41/47) and 85.1%(40/47) respectively.TrkA and VEGFR2 were expressed to different extents in SACC tissues of different pathological types,perineural invasion,et al.Significant correlation was been found in microvessel density(MVD) and VEGFR2 expression,P＜0.05;MVD in recrudescence group(26.58±2.38) was significantly higher than non-recrudescence group(19.06±1.39) in ACC tissues (P＜0.05);MVD in perineural invasion group(25.14±2.83) was significantly higher than non-perineural invasion group(18.81±1.33) in ACC tissues (P＜0.05)Conclusion:(1) The prepared shRNA targeting NGF,VEGF gene are capable in inhibitting the protein expression and mRNA transcription of NGF and VEGF in ACC cells.(2)TrkA and VEGFR2 play important roles in the invasion and metastasis of SACC.It is possible that TrkA and VEGFR2 could be chosen to evaluate the prognosis of SACC patients.