Study On The Molecular Mechanisms Of VEGF-C Responsible For Invasion And Metastasis And Reversal Strategy In Cervical Carcinoma

It is well known that Cervical carcinoma is one of the most common neoplastic diseases affecting women, with a combined worldwide incidence of almost half a million new cases annually,second only to breast cancer, it may be invasion and metastasis spreading via lymph node in the early stage . Despite there being sufficient evidence for the effectiveness of screening by cytology in preventing cancer of the cervix uteri, and incidence is increasing in younger women.It has been reported that VEGF-C may play an important role in cervical carcinoma and correlated with enhanced lymphatic metastasis.In our study ,to investigate the molecular mechanism of VEGF-C responsible for invasion and metastasis by immunohistochemistry ,transfection and RNAi so that to established experiment foundation in clinical therapeutic in the future.Part 1Expression of vascular endothelial growth factor-C andMMP9 TIMP-1 and clinical significance in cervical carcinoma.Objective To investigate the expression of vascular endothelial growth factor-c and MMP9 TIMP-1 in cervical carcinoma tissues and the relationship between cervical carcinoma clinicopathologic parameters. Methods The expression of VEGF-C, MMP9 and TIMP-1 examined in 62 cervical carcinoma tissues by immunohistochemistry technique(SP) and was statistically analyzed . Results There are higher expression about VEGF-C (77.4%) ,MMP9 (69.4%) ,TIMP-1 (62.9%) in cervical squamous cancer(I-II stage). Amongthem ,the expression rate of VEGF-C in I stage, II stage and tumor cells invasion lymph node is 73.8% 85%, 100.0% respectively;and the expression rate of MMP9 is 66.7%, 75% 93.3% respectively;TIMP-l is 64.3%, 60% 66.7% respectively. The expression of VEGF-C, MMP9 and TIMP-1 were higher in ≥1/2 depth of infiltration stromal than that of < 1/2 in cervical carcinoma tissues. The expression rate of VEGF-C MMP9 and TIMP-1 has the relationship with cancer cells in depth of infiltration and lymph node status . Conclusions Our research indicated that VEGF-C was relationship with tumor cells invasion and metastasis;VEGF-C, MMP9 and TIMP-1 play a important role in the development and aggressiveness of human cervical carcinoma.Part 2The effect of VEGF-C gene transfection on expression of VEGF-C in human cervical carcinoma cell HeLa and its molecularmechnisms of invasion and metastasis.Objective To explore the effect of VEGF-C gene transfection on the expression in the human cervical carcinoma cells HeLa. Methods An encoded fragment VEGF-C cDNA part of CDS was cloned from MDA-MB231 by RT-PCR. To construct the recombinant pcDNA3.1(+)/VEGF-C, a eukaryotic expression vector . The pcDNA3.1(+)/VEGF-C was transformed into human cervical cancer cells HeLa induced by liposome and was selected by G418.The changes in the expression level of VEGF-C mRNA and protein were determined by semi-quantitive RT-PCR and ELISA. Overexpression VEGF-C HeLa was named as HeLa /SI.The expression level of NF-kB ? bcl-2, MMP2, MMP9 , TIMP-1 mRNA were determined by RT-PCR in transfected cell lines. The sensitivity on cisplatin of the HeLa /SI was determined by MTT assay. The invasion and adhesion of the cell lines HeLa/Sl were analysis by Transwell filter. Results 1 An encoded fragment VEGF-C cDNApart of CDS was successfully cloned from MDA-MB231.The pcDNA3.1(+)/VEGF-C, a eukaryotic expression vector for human VEGF-C,is constructed successfully and identified by sequencing. 2 After transfection by liposome ,the VEGF-C mRNA level and the expression of VEGF-C protein in transfection HeLa cell lines were higher than those of control groups, respectively . Overexpression VEGF-C of HeLa/Sl cell lines is constructed successfully. 3 In HeLa/Sl cell lines the expression NF-kB gene (2.06 ±0.09 vs 1.35+0.02 vs 1.38±0.02 P <0. 05), bcl-2 gene mRNA(2.02±0.67 vs 0.41+0.06 vs 0.37+0.06 P<0. 05) level were also higher than those of control groups. 4 In HeLa/Sl cell lines the expression MMP2 gene (2.41 +0.81 vs 0.48±0.09 vs 0.49 +0.18 P<0. 05), MMP9 (2.26+0.29 vs 0.93+0.24 vs 0.79+0.05 /<0.05) gene mRNA level were higher than those of control groups, but TIMP-1 (0.54± 0.03 vs 1.01+0.06 vs 1.03+0.06 P<0. 05) was lower than those of control groups, the ratio of MMP9/TIMP-1 was higher than 1. 5 The sensitivity of HeLa/Sl on cisplatin was 1.84. 6 The invasion, metastasis and adhesion of HeLa/Sl were enhanced than those of control groups in vitro. Conclusions After VEGF-C gene transfection by liposome ,the expression level of VEGF-C in human cervical cancer cell lines HeLa/Sl is greatly heightened. The expression of MMP2. MMP9 gene in HeLa/Sl were also increased and TIMP-1 was lower than those of control groups;NF-kB was also stimulated and induced overexpression bcl-2 gene in HeLa/Sl by VEGF-C. To promote invasion and metastasis and adhesion in the HeLa/Sl phenotype.Part 3Inhibition of VEGF-C in HeLa cell lines by RNA interferencein vitro.Objective To study the growth inhibition of tumor cells by inhibiting VEGF-C expression of tumor cells with RNA interference (RNAi) technique, toanalysis the expression of VEGF-C* NF-kB * bcl-2 gene and the sensitivity on cisplatin of HeLa cell lines treated with VEGF-C siRNA. Methods According to the encoding sequence mRNA of human VEGF-C,the target site for RNAi was designed, siRNA was transfected by liposome and the expression of VEGF-C * NF-kBn bcl-2 mRNA was further observed by RT-PCR, to investigate the sensitivity of HeLa cell lines treated with siRNA on cisplatin by MTT analysis . Results The expression of VEGF-C of HeLa cell lines treated with siRNA 24h> 48h was significantly inhibited ,in 24h 80.63±0.24% ( P<0.001) and in 48h 38.9±0.85% (P < 0. 01 ) inhibited respectively;the expression of NF-kB was also down-regulated in 241k 48h, in 24h 37.55±2.76 % (P<0.05) and in 48h 30.5±3.82%( P=0.056) inhibited respectively;so that the expression of bcl-2 was also inhibited in 24h s 48h, in 24h 76.95±1.91% (P<0.01) and in 48h 64.11±2.96%( P<0.05) inhibited respectively. The sensitivity of the cells treated with siRNA on cisplatin was also enhanced about 40 fold . Conclusions After treated with VEGF-C siRNA of the HeLa cells,the expression of VEGF-C is effectively inhibited,NF-icB may be down-regulated and anti-apoptosis bcl-2 gene is effectively inhibited, so that the sensitivity on cisplatin of HeLa cell lines increased.