Expression Profiling And Identification Of Genes Associated With Lymphangiogenesis In Lung Adenocarcinoma

BackgroundLymphangiogenesis was not only involved in the lymphatic vessels of em bryonicdevelopment, tissue repair, chronic inflammation, but also in tumor lymph node metastasis.Metastatic tumor spread through lymphatic vessels occurs in lung carcinoma, with regionallymph-node metastasis often being the most important prognostic fac tor for carcinomapatients. Vascular endothelial growth factor (VEGF) -C, vascular endothelial growth factor(VEGF)-D, fibroblast growth factor (FGF) and platelet -derived growth factor (PDGF) arepotent secreted activators critical for tumor -induced lymphangiogenesis.High expression of VEGF-C and VEGF-D were confirmed in clinicopathological study,such non-small cell lung cancer (NSCLC), breast cancer, colon cancer, malignantmelanoma, head and neck cancer and prostate cancer, furthermore lymphatic microve sseldensity (LVD) and lymphatic vessel invasion (LVI) were closely related to lymph nodemetastasis. There were some specific molecular markers of lymphatic endothelial cells(LEC), such as (VEGFR-3), Prox-1, LYVE-1, Podoplanin and so on. With LEC isolationand establishment of lymphangiogenesis animal model, lymphangiogenesis study becomesa hot spot in tumor research. Our recent study showed that tumor cells overexpressingVEGF-C could induce lymphangiogenesis surrounding tumor cells and invasion oflymphatic vessels was a key step in the metastasis of primary tumors to draining lymphnodes.Although lymph node metastasis occurs frequently in lung adenocarcinoma, size ofprimary tumor and tumor occurrence of distant metastasis is not parallel in many cliniccases. We have reviewed mechanism of induction of lymphangiogenesis by tumor cells. Arethere other lymphangiogenesis promoting factors besides VEGF-C and VEGF-D? On theother hand, are there direct inhibitory lymphangiogenesis factors? Based on this hypothesis,we aimed at screening genes related with tumor lymphangiogenesiss. This research shows new approach for the lymphangiogenesis in lung adenocarcinoma and thus provides a newtarget for the inhibition of the metastasis of this kind of malignant car cinoma.ObjectiveTo Screen genes related lymphangiogenesis in lung adenocarcinoma by HumanGenome U133 Plus 2.0 Array, identify the selected genes for further experiment in vivo andin vitro based on bioinformatics analysis and fu nction reported in the literatures.Methods1. Podoplanin expression in 34 NSCLC tissues and 5 control benign pulmonary lesiontissues were detected by immunohistochemistry method. LVD score was determinedbetween LVDlow vs LVDhigh group, furthermore, the relationship LVD between tumorstaging, differentiation and pathologic N staging were analysesd.2. A high and low lymphangiogenesis in lung adenocarcinoma differentially expressedgenes cDNA library was constructed. Human Genome U133 Plus 2.0 Array was employedto screen differential expressed genes related to lymphangiogenesis in lung adenocarcinoma .Then differential expressed level was confirmed by real time PCR.3. IGFBP7 and CDH1 expression were determined with immunohistochemistry , aswell as IGFBP7 and CDH1 expression relat ionship was anlysesed with patient age, sex,tumor staging, differentiation degree and lymph node metastasis.4. RT-PCR detected expression of IGFBP7 in human lung cancer cell lines and mouselewis lung carcinoma cell (LLC).5. An interference plasmid, pGenesil-siIGFBP7 for a short hairpin siRNA directedIGFBP7 was constructed using the pGenesil -1 vector, and the effect of IGFBP7 knockdownthrough transfection the pGenesil-siIGFBP7 to LLC cells.6. Mice lymphangiomas in abdominal cavity were induced by intr aperitoneal injectionof incomplete Freund's adjuvant (IFA). LEC were obtained from the inducedlymphangiomas after disruption and digestion, and then were cultured in the flask or platepreviously coated with self-made rat-tail collagen. The capability of LEC to form lymphaticvessel-like structures was assessed by the in vitro lymphatic vessel formation assay.7. Transwell experiment and CCK-8 assay were used to determine whether IGFBP7knockdown affects cell migration of LLC cells and cell growth, respectively.8. The tumor models of IGFBP7 knockdown were established via subcutaneous injection of LLC cells in the dorsal site of the right ear of C57BL/6 mice, andlymphangiogenesis, lymph node metastasis and lung metastasis in vivo was investigated.Immunofluorence assay and confocal microscopy was used to examine the neo-lymphaticvessels.Results1. Podoplanin positive LVD was significantly higher peritumoral LVD ( 23.5±8.4) thanthat in benign pulmonary lesion (10.8±4.3) (P <0.01), and significantly higher thanintratumoral LVD (11.2±5.0). There is a statistical significance between lymph nodemetastasis N1-2 (24.3±8.7) and N0 (18.4±6.3) (P <0.01), as well as tumor stages (P <0.01).2. By the LVD scoring, 94 promoting lymphangiogenesis genes and 81 inhibitinggenes were screening. We repeated real time PCR to conform IGFBP7 and CDH1 genelevel.3. In 97 cases of NSCLC patients, positive expression of IGFBP7 was 54 cases,peritumoral LVD (23.1±8.5); negative 43 cases, peritumoral LVD (16.9±6.0). IGFBP7expression of tumor-positive LVD was significantly higher than negative tumor LVD ( P<0.05). Lymph node-positive group IGFBP7 positive rate (45.2%, 28/62) was significantlyhigher than node-negative group (25.7%, 9/35). But there was no significant associati onbetween CDH1 expression and lymph node metastasis, tumor stages, sex, age, histologicalclassification and differentiation stages ( P >0.05) was observed.4. RT-PCR detection of human lung cancer cell lines and mouse lewis lung carcinomacell over-expressed IGFBP7 gene, not detection in NIH3T3 and L929 cell lines.5. The vector pGenesil-siIGFBP7 targeting IGFBP7 was successfully constructed.6. Mice lymphangiomas in abdominal cavity were induced successfully by IFA, andmore than 96% living LEC were harve sted. The expression of VEGFR-3 was positive inLEC. In the gel formed by rat-tail collagen, LEC could form lymphatic vessel -likestructures.7. After the outer surface of the transwell insert of the LEC was found by HE staining,LLC group of cell membrane permeability was significantly higher than the percentage ofLLCSiRNA-IGFBP7 group (P <0.05); LLCSiRNA-IGFBP7 percentage was significantly higherthan the control group and the L929 group ( P <0.05). The results show that, IGFBP7knocked-down LLC can significantly enhance the capacity of LEC migration, but its capacity is less than the induction by not knock-down LLC. Immunofluorescence andconfocal microscopy showed that new -born lymphatic vessel is located peritumor and theirstructures were incomplete.8. At 45 days after tumor inoculation, the presence of supracervical node andcontralateral supracervical node metastases in control mice was 100% 33.3% and 66.7% inIGFBP7 knocked-down LLC inoculation, respectively, compared with nonfectcedgroup100% 83.3% and 100%; pGenesil-s1 group 100% 66.7 83.3%. In addition, themetastatic ratio of brachial nodes in IGFBP7 knocked-down tumors was significantly lowerthan that in control nonfiction and pGenesil -1 tumors. Immunochemical analysis forVEGFR3 expression LVD (9.4±2.3) was lower than that in control tumors was ( 16.7±3.5)and pGenesil-1(13.2±2.8) (P<0.05).Conclusions1. Human Genome Microarray was employed for comparison of expression profiles ofLVDhigh lung adenocarcinoma and LVDlow lung adenocarcinoma. IGFBP7 and CDH1 wereselected for further tumor lymphangiogesis study.2. IGFBP7 positive expression was correlated with tumor metastasis in lungadenocarcinoma, inducing LEC proliferation, migration and tube -like structure formation inLLC inoculated mice model, which indicating IGFBP7 should promote lymph nodemetastasis. CDH1 was not correlated with tumor lymphatic metastasis.