| BACKGROUND:Cholangiocarcinoma is an uncommonly malign-nant neoplasm in digestive tract, and accounts for about2%～3%of all digestive tract cancers. This cancer was described firstly by Durand Fardel in1840, which was originating from the biliary epithelium (cholangiocytes), and was most usually in the tumor of liver and biliary tract system, the incidence rate reached10%～15%in the liver and biliary cancer. The incidence rate of this disease was about1/100,000in the worldwide, but in the near decade this rate tended to increase. In our country, this rate of increase account to5%every year, and this increase rate of development became to the fastest in the all of digestive tract cancers. Therapeutic approach as well as surgical technology, radiothe-rapy, chemotherapy, and immunity treatment advanced more than imagine. Because of especially pathological features and exceptive anatomical position for this tumor, the patient with cholangiocarcinoma lack of specific clinical symptoms and diagnostic technology on the early stage. When many patients appear clinical symptoms, the ill was on the progression or advanced stage, due to they have no chance to achieve radical operation. So that the3-year survival rate only was35%to50%on the post surgery, and the5-year survival rate less than10%worldwide, as well as5%in our country. Due to the diagnostic level and therapeutic efficacy have no conspicuously improved, so these neoplasms extremely threaten the health of people. For those reasons, it’s vital significance to further investigation of mechanisms of cholangiocarcinoma carcinogen-nesis, progression, invasion and metastasis. Therefore, it is current research focus to understand cholangiocarcinoma pathogenesis in the molecular biology, and to seek the targets of malignant transformation of cholangiocarcinoma, as well as targeted therapy of this tumor.Vascular endothelial growth factor (VEGF) takes part in the most all of aspects of vessel and lymphatic formation. It was generally ackno-wledged a specificity factor, which was one of the most effectively and strongly to neo-angiogenesis of tumors. VEGF has been shown to make a most important contribution for the angiogenesis of a solid tumor and its metastasis, moreover to take an important initiation factor for angio-genesis.MMP-2gene was one of the most important and widely distributed members in the matrix metalloproteinase (MMPs) family. Its functions were to depredate extracellular matrix (ECM), to destroy the histological barriers in the process of invasion, and then to lead tumor cells breakthrough ECM and basilar membrane, infiltrate to closed fibrous connective tissue, take place in the metastasis. It plays a key role on the invasion and metastasis to tumor cells.RNA interference (RNAi) is the classic kind of post-transcriptional gene silencing method, which allows to specifically blocking the expres-sing of target genes at the mRNA level, so it have most of specificity and effectivity. RNAi is now being exploited as a powerful tool for reverse genetics, widely powerful tool for the analysis of gene function and shows great promise for therapeutic applications.OBJECTIVE:In part one, to investigate the expression of VEGF-A, VEGF-C and MMP-2in the samples of cholangiocarcinoma tissues and analyze the relationship between expression of VEGF-A, VEGF-C, MMP-2and clinicopathological features, prognosis of cholangiocar-cinoma. In order to further explain the functions of VEGF-A, VEGF-C and MMP-2protein in the process of carcinogenesis, invasion, metastasis of cholangiocarcinoma, we detect the microvascular density (MVD) and micro lymphatic vessel density (MLVD) in the samples of cholangiocar-cinoma tissues. In part two, we successfully design and synthesize siRNA sequences of targeting VEGF gene, and chose a most effective siRNA to the next experiments. In part three, we use VEGF-siRNA to down-regu-late the expression of VEGF-A, VEGF-C and MMP-2gene in QBC939cell line, in order to explore the molecular mechanism of these3genes in the angiogenesis, proliferation, invasion and metastasis of cholangiocar-cinoma.METHODS:1. The expression of VEGF-A, VEGF-C and MMP-2in47specimens of cholangiocarcinoma tissues,40adjacent non-cancer specimens and15normal biliary tissues was detected by immunohistochemistry. The relationship between expression of VEGF-A, VEGF-C, MMP-2and clinicopathological features, prognosis was statistically analyzed.2. We used CD105and D2-40to mark the vascular and lymphatic endothelial cells in47cholangiocarcinoma tissues specimens,40adjacent non-cancer specimens and15normal biliary tissues, respectively. And then to count the number of MVD and MLVD under microscope, in order to analyze the relationship between MVD, MLVD and VEGF-A, VEGF-C, MMP-2, so that further explain the function of VEGF-A, VEGF-C and MMP-2protein in the process of carcinogenesis, invasion, metastasis of cholangiocarcinoma.3. We designed and synthesized siRNA sequences of targeting VEGF gene, cholangiocarcinoma cell line QBC939, HCCC-9810and RBE were respectively transfected with VEGF-siRNA plasmids for48h by LipofectamineTM2000. After the transfection, we measured the inhibited efficiency in VEGF expression; choose the most efficient one and QBC939cell line for further study.4. After the transfection, the RT-PCR and Western blotting test were used to measure the expression levels of mRNA and protein of VEGF-A, VEGF-C and MMP2in the QBC939cell line, respectively.5. The cell invasion potential was preformed by Transwell invasion and migration assay, and MTT assay was employed to detect the proliferation of the cholangiocarcinoma cell. Flow cytometry was employed to evaluate cell apoptosis and death.RESULTS:1. The expression of VEGF-A, VEGF-C and MMP-2proteins in cholangiocarcinoma tissues was significantly higher than that in adjacent non-cancer tissues and normal biliary tissues (P<0.01). Overexpression of VEGF-A, VEGF-C was statistically significantly associated with the depth of invasion, TNM stage and lymphatic metastasis. Overexpression of MMP-2was statistically significantly associated with the depth of invasion, differentiation degree, TNM stage and lymphatic metastasis. There are positive correlation between the expressions of VEGF-A, VEGF-C and MMP-2on protein level (P<0.05). In Cox multivariate analysis, expression of VEGF-A, VEGF-C and MMP-2immunostaining is found to be independent prognostic factors.2. MVD in cholangiocarcinoma tissues was significantly higher than that in adjacent non-cancer tissues and normal biliary tissues (P<0.01); as well as MLVD in cholangiocarcinoma tissues and adjacent non-cancer tissues both significantly higher than that in normal biliary tissues (P<0.01). The number of MVD and MLVD was statistically significantly associated with the depth of invasion, differentiation degree, TNM stage and lymphatic metastasis. There are positive correlation between the expressions of VEGF-A, MMP-2and MVD; and these three parameters can reflect the status of neo-angiogenesis in cholangiocarcinoma. There are positive correlation between the expressions of VEGF-C, MMP-2and MLVD; and these three parameters can reflect the status of neo-lymphan-giogenesis in cholangiocarcinoma.3. We designed and synthesized siRNA sequences of targeting VEGF gene, cholangiocarcinoma cell line QBC939, HCCC-9810and RBE were successfully transfected with VEGF-siRNA plasmids, respecti-vely. Compared to negative control group and blank control group, the mRNA and protein level of VEGF in VEGF-siRNA-1group were reduced by86.37%and79.72%, respectively.4. After the transfection of VEGF-siRNA-1, we can observe both the mRNA and protein of VEGF-A, VEGF-C and MMP2were signify-cantly down-regulated in the QBC939cell line, whereas the invasion, migration and proliferation of tumor cells were decreased greatly. The rate of apoptosis of tumor cells was increased obviously in the VEGF-siRNA group compared with the blank control and negative control group (P<0.01).CONCLUSION1. VEGF-A, VEGF-C and MMP-2are overexpression in the cholangiocarcinoma tissues, and maybe have synergetic effect in the process of carcinogenesis, invasion, and metastasis of cholangiocar-cinoma.2. Overexpression of VEGF-A, VEGF-C and MMP-2have significantly associated with the depth of invasion, differentiation degree, TNM stage and lymphatic metastasis in the cholangiocarcinoma.3. VEGF-A, VEGF-C and MMP-2involve within the regulation of MVD and MLVD level in the cholangiocarcinoma, and they have significant positive correlation. Moreover, they make an important contribution to the angiogenesis and lymphangiogenesis in the cholangiocarcinoma.4. VEGF-siRNA can efficiently and specifically inhibit expression of VEGF gene on mRNA and protein level in cholangiocarcinoma cell line QBC939, HCCC-9810and RBE, respectively.5. When VEGF gene was silenced, we can observe both the mRNA and protein of VEGF-A, VEGF-C and MMP2were significantly down-regulated in the QBC939cell line.6. Blocking the expression of VEGF gene via VEGF-siRNA can effectively inhibit the invasion, migration, proliferation, and induce apoptosis in the QBC939cell line. These findings suggest that the RNAi approach targeting VEGF maybe an effective therapeutic strategy for cholangiocarcinoma and provide a new idea for the treatment.7. The expressions of VEGF-A, VEGF-C and MMP-2on mRNA and protein level have significantly associated with carcinogenesis, progression, and prognosis of cholangiocarcinoma. In addition, detection and administration the serum level associated with VEGF-A, VEGF-C, MMP2, MVD and MLVD maybe a useful and significant prognostic indicator. |
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