The Expression Of Vascular Endothelial Growth Factor-C And The Regulation By RNAi In Cervical Carcer

PartⅠEXPRESSIONS OF VASCULAR ENDOTHELIAL GROWTH FACTOR-C IN CERVICAL CANCER AND ITS CLINICOPATHOLOGICAL SIGNIFICANCEObjective To investigate the expression of vascular endothelial growth factor C(VEGF-C) mRNA in cervical cancer tissues /para-tumour and normal cervix tissues.The relationships between related mRNAs expression and the tumor metastasis,prognosis were analyzed.Methods Cervical cancer tissues(n=48),para-tumour tissue(n=48) and their normal controls(n=36)were collected.Using TaqMan real-time fluorescent quantitative PCR method to detect the expressions of VEGF-C mRNA,To analyze the conjunction of the factors' expressions with the clinical features of the tumor.Results Through real-time fluorescent quantitative PCR method,we funnd that there was a significant difference levels of VEGF-CmRNA expression between the tissues of tumor,para-tumour tissue and that of the normal controls(P＜0.05). The levels of VEGF-C mRNA expression in invasive carcinoma of cervix was higher than that in all matched.And there was no significant relationship between the levels of VEGF-C mRNA expression with the clinical stagein invasive carcinoma of cervix (P＞0.05).But the levels of VEGF-C mRNA expression was significant associated with the pathological types,the grade of tumor pathology,lymph node metastasis, tumor size and the invasion of deep muscular layer(P＜0.05).Conclusion The method of detection for the VEGF-C mRNA expression in cervical cancer tissues by real-time fluorescent quantitative RT-PCR has a high sensitivity and specificity.It is reliable and easily performed.The results suggest that the VEGF-C may play an important role in genesis and development of invasive carcinoma of cervix.It would be a useful biological marker to foresee the lymph node metastasis and monitor the progression of invasive carcinoma of cervix.VEGF-C might be used as a potential molecular target for the treatment of malignant tumors. PartⅡTO DETECT THE EXPRESSION OF VASCULAR ENDOTHELIAL GROWTH FACTOR C mRNA IN CERVICAL CANCER CELL LINES HELA CELLS AND SiHa CELLS BY REAL-TIME QUANTITATIVEPCRObjective To investigate the relationship of vascular endothelial growth factor C and the growth and proliferation of cervical cancer.Methods Cervical cancer cell lines,Hela cell lines and SiHa cell lines,were cultured and detected the contents of VEGF-C mRNA by TaqMan real-time fluorescent quantitative RT-PCR expression.Results The levels of VEGF-C mRNA expression in Hela cell lines was higher significantly than that in SiHa cell line(t=7.1,P＜0.05).Conclusions VEGF-C gene may be related to ability of invasion of cervical cancer,inhibiting the expression of VEGF-C is expected to has the function of inhibiting the invasion and metastasis of cervical cancer. PartⅢTHE EFFECTS OF SiRNA TARGET VEGF-C ON BOTH THE EXPRESSION OF VEGF-C AND THE CELLS PROLIFERATION OF CERVICAL CANCERObjective To study the effects of small interference RNA on VEGF-C mRNA expression in vitro on cultured Hela cell lines and Siha cell lines,the observation of proliferation of the cervical cancer cells,and the exploration of the application of RNA interference mediated by VEGFC-siRNA on cervical cancer gene therapy in the future.Methods On the basis of the principle of target sequence of siRNA,three interference sequences of VEGF-C were designed.The best siRNA interference sequences was screened and then transfected into VEGF-C overexpressed cervical cancer cell line,Hela cells in vitro,and VEGF-C low-expressed cervical cancer cell line,Siha cells in vitro,by siRNAs-liposome complex.VEGF-C mRNA level was detected by real time RT-PCR after transfection by siRNA and began to observe and measure the results of RNA interference in 12 hours.The VEGF-C protein was analyzed by Western blot between transfection and controls.MTT method and cell count methods were used to observe the effects of RNAi on the bioactive behalves of cells.Results After transfected,the cells in the experimental group,deformate, shrink,and began falling out.The growth of Hela cells and Siha cells were both obviously inhibited,Whereas the cells in control groups had not obviously changed. Sequence specific siRNAs of VEGF-C effectively downregulated the mRNA level of VEGF-C.Western blot revealed that the protein levels were down regulated obviously post transfection compared with the control in 12h/24h/48h and 72h(P＜0.05).They were significantly different compared with the control groups.During 12h-72h after transfected,the inhibition ratio of proliferation in both Hela cells and Siha cells were obviously higher than the controls'(P＜0.05).There was a significant difference in the levels of cell population of experimental group and the controls during 12h-72h(P＜0.05).Conclusions The small interference RNA target VEGF-C can effectively downregulate VEGF-C on both mRNA and protein,inhibit the growth of Hela cells and Siha cells.It hints that VEGF-C may plays an important role in the development of cervical cancer,which provides an experimental basis for treating human tumors with anti-angiogenesis method using RNAi..