The Experimental Research Of The Expression Of CCBE1 In Lung Cancer And The Relationship Between CCBE1 And VEGF-C

Backgroud:Lymphatic metastasis is one of the most common ways to lung cancer, the existence of lymph node metastasis is an important factor affecting for the prognosis of lung cancer. Currently anti-lymphatic is the new direction of cancer treatment. Ithas been clearly formed that tumor-associated lymphatic pathway is necessary for lymphatic metastasis of tumor cells, but the mechanism of tumor and lymph node lymphangiogenesis is still unclear. Researches, by blocking vascular endothelial growth factor receptor-3 signal to inhibitlymphangiogenesis, or by neutralizing chemokines (eg SLC/CCL21) to inhibit lymph node metastasis in animal models, suggested that lymphangiogenesis and lymphatic tumor cells, lymph node metastasis can happen through different pathways liketumor angiogenesis, in which involved a variety of regular machanisms. Revealing thelymphatic vessel formation mechanism before the lymphatic metastasis of tumor cells may contributes to proposing new anti-metastatic strategies. 1)endothelial progenitor cellsand lymphangiogenesisLymphangiogenesis is essential for lymph node metastasis andprognosis. Previous studies suggest that tumor tissue can not form lymphatic vessels, but this situation cannot explain tumor lymphatic metastasis,recent studies have found dispersed or bundled of lymphatic in tumor tissue or around the tumor and the densityof lymphatic in tumor is more intensive than normal lymphatic tissue. The experiments of Mandriota have discovered that the solid tumor can formed lymphatic vessels.Endothelial progenitor cells are precursor cells of endothelial cells. In 1997, Asahara and his fellows, being the first group, isolated the vascular EPCs from human peripheral blood concluding CD34+/VEGFR-3+that can differentiate into vascular endothelial cells. Vascular EPCs, which located in the bone marrow after birth,under the influence of cytokines, can be moved from the bone marrow into the peripheral blood circulation mobilization toform local tissue angiogenesis in pathological conditions such asblood vessel injury, tissue ischemia and tumorigenesis. Salyen has isolated cells concluding CD34+/CD 133+/VEGFR-3+from fetal liver in 2003. This kind of cells could differentiate into lymphatic endothelial cells or endothelial cells. In 2005, Wang have isolated cells from umbilical cord blood concluding CD34 +/CD 133+/VEGFR-3+logo EPCs, which could be induced by VEGF-C to differentiation into lymphatic endothelial cells, so the CD34+/CD 133+/VEGFR-3 +EPCs was named lymphatic endothelial progenitor cells to distinguishedwith the vascular EPCs. The study have shown that LEPCs can form the lymphangiogenesis in vivosuch as renal transplantation. Data showed that peripheral blood VEGFR-3+ LEPCs strong positive in non-small cell lung cancer patients are with poor prognosis, it may be related to an earlier occurrence of lymph vessels, lymph nodes micro-metastasis.2) SDF-1/CXCR4 andlymphangiogenesisIn recent years, the interection of CXC chemokine receptor-4 and its ligand stromal cell-derived factor-1 to form reaction is drawing people’s attention. It may be related to the induction of tumor angiogenesis and regulated tumor angiogenesis. The SDF-1/CXCR4 axis drought appears in breast cancer research.2001 Muller discovered human breast cancer cell lines, primary breast cancer tumor and metastases with high expression of chemokine receptor CXCR4 and CCR7, and even the most common metastases of breast cancer to lymph nodes, lungs, liver and bone marrow had a high level expression of its ligand SDF-1 and CCL21, thus,a high expression of CXCR in early breast cancer may presagea high risk of recurrence. More and more studies showed that a variety of solid tumors,including non-small cell lung cancer, which is closely related to differences in their metastatic potential of the expression of CXCR4, have an excess of expression of CXCR4. A recent study found that SDF-1/CXCR4, by activatingthe signaling system of ERK, activates IKK and NF-kB signal pathway to increase the expression of MMM-9, and promote migration of lung cancer cells. The SDF-1/CXCR4 axis is primarily related to angiogenesis. Cancer cell by clone proliferation to invade local tissue by vascular endothelial growth factor to induce angiogenesis and have a high expression of CXCR4, then the cancer cells pass through the angiogenesis and are captured by rich in SDF-1 in the blood vessel wall concluding CD34+cells, can complete the transfer from cancer tissueto normal tissue. Subsequent studies found that the expression of CXCR have correlated with lymph node metastasis. Kato, by analyzing 79 cases of surgical resection of Infiltrating ductal breast cancer, have detectedthat breast cancer that through the lymphatic metastasis is also accompanied by high expression of CXCR4. Tanabe found that the expression levels of CXCR4mRNA had positively correlated with cat breast lymphatic invasion in animal experiments. But theusage of CXCR4 antagonist T140 or antisense RNA interference can inhibit migration of breast cancer cells in vitro reaction. Angiogenesis and lymphangiogenesis have the same pre-progenitor cells, the SDF-1/CXCR can regulate angiogenesis. What can SDF-1/ CXCRaxis work in the lymphangiogenesis of lung cancer?3) VEGF-C/VEGFR-3 and lymphatic metastasis of lung cancerVEGF-C, which belongs to the VEGF family, was the most specific lymphatic endothelial at present, and VEGFR-3 that has a high chemical affinity with lymphatic epithelium and can induce proliferation of lymphoid and lymphangiogenesis, ever has a high expression in rich of lymphatic is its specific receptors. In non-small cell lung cancer patients with lymph node metastasis has higher level of VEGF-C than those without lymph node metastasis. There is a significant correlation between the expression intensity of VEGF-C/VEGFR-3 and the lymphangiogenesis and lymph node metastasis. VEGF-C as a ligandto bind with its acceptor VEGFR-3 to phosphorylate the receptor,and then to play tyrosine kinase activity, which using MEK/ERK and PI3/AKT pathway to stimulate lymphatic endothelial cell mitosis, promote lymphangiogenesis and make use of lymphangiogenesis factors. If theactivation of SDF-1/CXCR can stimulate lymphatic in lung cancer, whether or not is it associated with the promotion of LEPCs to expression of VEGF-C?4) CCBE1 and VEGF-C and lymphatic metastasisExperiments confirmed that CCBE1 plays a vital role in the activation of VEGF-C. Phenotypic analysis of murine Ccbel mutant embryos showed a complete lack ofdefinitive lymphatic structures, even though Proxl+lymphatic endothelial cells that specified within the cardinalvein cannot form this structure. Mutant mice die prenatally. Thisexperiment shows that VEGF-C/VEGFR-3 pathway activation of the lymphatic system can be achieved only before CCBE1 mutation. Therefore, CCBE1 in the embryonic period by inducing VEGF-C to make it active, thus contributing to the lymphangiogenesis. However, there are experiments confirmed that CCBE1 has little effect for generating lymphatic, itmainly through the activation of VEGF-C/ VEGFR-3 pathway to get work for lymphangiogenesis. Thus, our data suggestCCBEl to be essential but not sufficient for lymphangiogenesis.Objective:1. By collecting human lung cancer tissue and normal tissue to do comparison,and then detecting the expression of the two groups of CCBE1 and the specific antibody LYVE-1 to confirm the expression of LYVE-1 between the two tissues, and then definitethe relationship between the CCBE1 and the LYVE-1;2. Introduce three groups of low virus into mouse lung cancer cells,after stable expression found in cells, add to puromycinto to kill the cancer cells which have no low virus in, then make the left cells as filter cells to use.3. using western blot to choose the best inhibitory effect of a virus strain for use;4. injectthe lung cancer cells with process and lung cancer cells without any process into mice,and then detect the expression of VEGF-C in both groups of mice, fininely, making conclusion between the two.Methods:1. Use Q-PCR technology to detect the expression of CCBE1 in normal tissue and lung cancer tissue; then use immunohistochemistry and western blot technique to detect the expression of CCBE1 and LYVE-1 in lung cancer and normal tissue.2. culturing mouse lung cancer cells under sterile conditions, at about the third generations with a stable culture and then transfected virus into cells and observe the state under a fluorescence microscope, and thenuse puromycin to choose;3.put the three groups of cells containing the virus under western blot detection; according to the results of western blot to select the group of the most obvious effect of suppressing, then injected the group of the most obvious effect of suppressing and untreated lung cancer cells into adult male body divided into two groups in advance. Until the time of tumor formation, sacrifice the mice, resectthe tumor tissues completely,then usewestern blot technologyto observe the expression of VEGF-C in two groups.Results:1. the expression of CCBE1 and its relationship with LYVE-1 in lung cancerIn the Q-PCR, there exist significant difference in normal tissues and in cancer tissues, the expression of CCBE1 in normal tissue is higher than that in lung cancer, especially tissues with lymph node metastasis shows lower expression of CCBE1, so we can concludedthat CCBE1 in lung cancer has low expression; in immunohistochemistry and western blot experiments, the expression of CCBE1 in normal tissues is higher than in lung cancer in the two tissues, which is coincide with the results of Q-PCR, but for the lymphatic specific labeled antibody--LYVE-1, it is contrary with the expression level of CCBE1, theexpression is higher than in normal tissue, it even has higher expression with lymph node metastasis. This result indicates that the expression of CCBE1 has a negative correlation with LYVE-1.2. virus into mouse lung cancer cellsPut the lung cancer cells prepared in advance in a temperature, and then injected virus together with polybrene witha appropriate percentage to preparation and use fluorescence microscopy to observe. Over time, the number of cells with virus increase and the number of fluorescent cells in the microscopic field is growing.3. choosing of the lung cancer cellsUse nutrient solution with puromycin to replace the original nutrient solution in lung cancer cells, and the lung cancer cells without the virus will be killed. Floating on the surface of the culture medium, use nutrient solution without puromycin after 12 hours until cells were grown to about 80% of the bottle, then do the second choosing.4. The best screening effect through inhibition of lung cancer cellsBy introducing the three groups of lung cancer cells with western blot,we can know that the ShRNA2 hasthe best inhibitory, and the other two also have a certain inhibitory, but not as good as the inhibitory of ShRNA2, according to the experimental results, ShRNA2 can be considered the best effect of suppressing.5.puttingthe best effect of suppressing in vivo into mouse and analysisAfter the tumor cells are completely resected out of the tumor tissue, use western blot to detect the expression of VEGF-C of the two groups, and the results showed that the expression of lung cancer group with process was higher than untreated cells.Conclusion:1. the expression of CCBE1 innormal tissue is higher than lung cancer, especially in lymph node metastasis of lung cancer tissue, the expression is lower; but the expression of LYVE-1 is opposite with the expression of CCBE1,which is higher in cancer tissue than normal tissue, even higher in cancer tisssues with lymph node metastasis.2. the expression of CCBE1 in lung cancer cells with the process of virus is lower compared with untreated cells expressed.3. CCBE1 can secrete a kind of protective protein, which can inhibit the secretion of VEGF-C protein, and thereby inhibiting lymphangiogenesis.