The Study Of Sema3F Signal By NRP2 Mediate To Effect Lymphangiogenesis And The Metastasis In Colorectal Carcinoma

Background and ObjectiveColorectal cancer,a common malignant tumor in digestive system,its metastasis is mainly through lymphatic vessels.Studies during the last few years have provided significant insights into the molecular mechanisms underlying the development of lymphatic vessels and the of lymphangiogenesis in health and disease.With the finding of the specific markers of lymphatic and lymphangiogenesis factors,they could become the next focus forlymphangiogenesis and metastasis research.Recent developments have highlighted the importance of lymphatic endothelial cells(LECs) and role of lymphangiogenesis in malignant tumor invasion and metastasis.Neuropilins are 130to 140-kD a single spanning transmembrane glycoproteins that function as receptors for semaphorins,which induces the collapse of neuronal growth cones. neuropilin-2(NRP2) is found to act as a receptor for sema-3F,which induces repulsion of NRP2 expressing neuronal growth cones.NRP2 has also been shown to bind several splice form of VEGF,including VEGF₁₆₅,VEGF₁₄₅,and VEGF-C,but not VEGF₁₂₁.These studies suggest that NRP2 acts as a co-receptor for VEGFs to inhibit angiogenesis.These two different ligand bind same receptor mediate two different process——neuronal development and angiogenesis,which suggest these process may be same molecular mechanisms.In the embryonic vasculature,NRP1 is expressed by vascular ECs whereas NRP2 is expressed by venous ECs and LECs.Which indicated NRP2 implicate with lymphangiogenesis.Homozygous Nrp2 mutants show absence or severe reduction of small lymphatic vessels and capillaries during development.This correlated with a reduction of DNAsynthesis in the lymphatic endothelial cells of the mutants.Arteries,veins and larger, collecting lymphatic vessels developed normally,suggesting that neuropilin2 is selectively required for the formation of small lymphatic vessels and capillaries.The first detectable lymphatic vessels in the embryo are the thoracic duct and jugular lymph sacs,which are thought to develop by sprouting from veins.NRP2 is likely to be downstream of these initial events in the formation of lymphatic ECs,NRP2 staining carried out at different stages of development showed a progressive coverage of formation of lymphatic ECs, Therefore,it may be speculated that NRP2 function is not required for sprouting of lymphatic vessels from veins,but for the sprouting of LECs from pre-existing lymphatic EC. These observations of staining suggest that NRP2 function is required for the formation lymphocapillary vessel from pre-existing lymphatic EC.However,duringing neural axon guidance process,sema3F bind NRP2 induced repel axons from growing neurons,not promote axons from growing neurons.There may be present the similar repel mode between sema3F bind NRP2 for colorectal LECs which expressing NRP2 and repel axons from growing neurons.It may block LECs chemotatic activity and inhibit tumor lymphangiogenesis.Therefore,which Signal of Sema3F by NRP2 mediated was presume to repress tumor lymphangiogenesis and tumor invasion and metastasis.Sema3F may be a potential inhibitor for tumor lymphangiogenesis and tumor metastasis.In this study we detected signal of Sema3F by NRP2 mediate to effect lymphangiogenesis and the colorectal carcinoma in order to explore its contribution in colon cancer lymphangiogenesis and lymphonode metastasis and its potetial mechaninsm, which may laid a foundation for further understanding the mechaninsm of lymphatic metastasis route in colon cancer.Methods1.Colon cancer tissue immunohistochemistry assayHuman colon cancer samples,obtained from southwest hospital surgery department, were sliced and detected the expression of Sema3F,and NRP2,andVEGF-C,and lymphantic density with immunohistochemical analysis.The results were analyzed statistically to investigate the correlations of among the expression of Sema3F,and NRP2 and VEGF-C with lymphonode metastasis,and lymphatic counts,and some other clinical pathology characterictics.2.Effect of Sema3F signal by NRP2 mediate to lymphangiogenesis and lymphonode metastasis in colon cancer assayTo constructe the NRP2 and VEGF-C genes RNA interference eukaryotic vector.After transfection Sema3F,and NRP2 RNAi,and VEGF-C RNAi vector into LOVO cells, simultaneouly,co-transfection Sema3F+ NRP2 RNAi or VEGF-C RNAi into colorectal LOVO cell lins,then transplanted into nude mouses.After harvested the xenotransplant neoplasm,the expression of Sema3F,and NRP2 and VEGF-C,and tumor cell apoptosis, and proliferation,and the lymphantic density,and lymphonode metastasis and distance organ metastasis in the xenotransplant tumor were dectected and compared with the control group.3.NRP2's effect on hLECs function in vitroTo isolate hLECs from foreskin derma by immunomagnetic beads.Then,the recombinant expression vector and RNAi vector of NRP2 was constructed and transfected into hLECs.To asssay differention of the migrate by Boyden chamber,chemorepulsant by coculture with LOVO cells and canaliculization by three-dimensional culture for hLECs with different NRP2 express level.Simultaneous,To asssay integrinα_Ⅴβ₃ expressing level change in hLECs with different NRP2 expressing level by western blotting and RT-PCR.Results1.The expreesion of podoplanin was location on cell membrane and cytoplasm of LECs in colorectal tumor tissu and colorectal normal tissu.There was not the significance different between MLD of colorectal tumors(29.5±12.2) and colonic normal tissu(25.4±8.0).The significant differention were among those of tumor margin(48.8±23.5), superficial tumor region(24.6±10.1) and tumor center(11.3±7.7)(P＜0.05);The major lymphatic vessels in the tumor margin were expand,however,it in superficial tumor region and tumor center were atretic trabs.The Ki-67 label index(LI) of LECs in colorectal tumors tissu was 0.17±0.11,but that in normal colonic tissu almost was zero.The Ki-67 LI of LECs was highest in tumor margin(0.22±0.12),that in central tumor region(0.12±0.08) was lowest,that in superficial tumor region was 0.15±0.10.Furthermore,the significant differention was between tumor margin and central tumor region(P＜0.05).2.The mRNA expression of NRP2 in tumor tissu(0.87±0.26) was higher than that(0.38±0.12) in normal tissu(p＜0.01);that(1.12±0.25)in tumor tisssu of metastasis group was higher than that(0.76±0.19) in non-metastasis group(p＜0.01),the expression of NRP2 were location on cytoplasm in tmor cell and LECs,it's expression rate was 34.8%.Their MLD were correlated with expression strength of NRP2(r=0.432,P＜0.01).The expression of NRP2 was association with lymphatic nod metastasis,and differentiation degree,and Duke's stage and infiltrate degree of tumors(P＜0.05).However,it was not association with patient's age,and patient's sex,and size of tumors,and location of tumors(P＞0.05).3.The mRNA expression of Sema3F in tumor tissu(1.26±0.17) was higher than that(1.17±0.15) in normal tissu(p＞0.05);that(1.20±0.16)in tumor tisssu of metastasis group was lower than that(1.38±0.22) in non-metastasis group(p＜0.05),the expression of Sema3F were major location on cytoplasm in tmor cell,but in some sample,it's expression was location in ellular nucleus of tmor cells and mesenchymal cells,it's expression rate was 50%.The MLD of Sema3F positive group was 21.8±10.2,and signifcance lower than that(33.5±12.6) of Sema3F negative group(p＜0.05).The expression of Sema3F was association with lymphatic nod metastasis,and differentiation degree,and Duke's stage and infiltrate degree of tumors(P＜0.05).However,it was not association with patient's age,and patient's sex,and size of tumors,and location of tumors(P＞0.05).4.The expression of Sema3F in colorectal cancer tissu was negative correlated with expression of NRP2(r=0.099,P=0.013).it was negative correlated with expression of VEGF-C(r=0.174,P=0.001).On the contrast,the expression of NRP2 in colorectal cancer tissu was positive correlated with expression of VEGF-C(r=0.606,P=0.001).5.To successfully construct the NRP2 and VEGF-C genes RNA interference eukaryotic expression vector,and successfully transfection them into LOVO cells.To compare with control test group,the inhibiton rate of NRP2and VEGF-C mRNA and potein expression of LOVO cells respectively were76.5%and 74.9%,77.3%,75%and 76.6%,74.5%.6.To successfully transfection the Sema3F gene vector and NRP2,VEGF-C RNAi vector to LOVO cells,Furthermore,to cotransfection the Sema3F+ NRP2 RNAi or VEGF-C RNAi vector to it.To compared the microlymphatic density in xenotransplant neoplasm of test group were significant lower than those in contral group at same transplante time(p＜0.05).All nude of contral group were find the distant metastasis.The nude were transplanted LOVO cell which merely suppressexpressing NRP2 or VEGF-C were find the lymphatic node and distant metastasis until 8 weeks.Furthermore,the nude were transplanted LOVO cell which overexpressing Sema3F and suppressexpressing NRP2 group had similar result,the nude were transplanted LOVO cell which only overexpressing Sema3F group were find the distant metastasis until 12 weeks.Howerve,the nude were transplanted LOVO cell which overexpressing Sema3F and suppressexpressing VEGF-C group were not find the distant metastasis until 12 weeks.On the otherhand,the glander structure were find in the xenotransplant neoplasm samples of nude were transplanted LOVO which overexpressing Sema3F groups,it indicated that Sema3F could induce the nonmetastatic tumor phenotype.Furthermore,that tumors expressing Sema3F contained large areas void of viable tumor cells.These areas were composed of massive numbers of TUNEL-positive apoptotic cells and lacked dividing cells.Control tumors,on the other hand,contained mainly actively dividing cells as identified by proliferating cell nuclear antigen(ki-67) staining and were nearly void of apoptotic cells.7.To successfully isolation and purify human hLECs from foreskin derma.It's expression of D2-40,VEGFR3,NRP2,podoplnain and LYV-1 were deteced by immunofluorescence,but notâ…§factor.The S and G₂/M phase of VEGF-C(10ng/ml) induce group of hLECs were signifcance increase than those of contrl group(p＜0.05).8.3.NRP2's effect on LECs function in vitroTo successfully transfection the NRP2 gene and it's RNAi vetor into hLECs.The capable of migrate,cell adhesion and canaliculization in hLECs which overexpressing NRP2 were significant higher than those in hLECs which expressing and down regulation expressing NRP2(p＜0.05).Furthermore,the overexpression NRP2 in hLECs induce integrinα_Ⅴβ_â…¢expressing level than those of hLECs which expressing and down regulation expressing NRP2(p＜0.05).On the contrast,the proliferation of hLECs had not difference among in different NRP2 expressing level hLECs.Furthermore,the LOVO which overexpressing Sema3F could induce chemical-repulsion to hLECs which expressing of NRP2,but the LOVO which overexpressing Sema3F could growth to mix together with hLECs which down regulation expressing NRP2.Conclusion1.The expression of Sema3F and VEGF-C and NRP2 in conlorectal cacer tissue is associated with lymphangiogenesis and the metastasis in colorectal carcinoma.The expression of Sema3F is inverse correlation with VEGF-C and NRP2 expreesion in colorectal cancer tissue.On the contrast,the expression of NRP2 is positive correlation with NRP2 expreesion in colorectal cancer tissue. 2.The NRP2 and VEGF-C RNAi eukaryotic expression vectors are successfully constructed.3.Sema3F could induce the nonmetastatic tumor phenotype.Furthermore,it could bind NRP2 to inhibit the lymphangiogenesis and the metastasis in colorectal carcinoma. There is a mechanism that Sema3F and VEGF-C completely bind co-receptor NRP2 to inhibited lymphangiogenesis of tumor.4.The human lymphatic endothelial cells is successfully isolated and purified and identified.Moreover,The human lymphatic endothelial cells three-dimensional culture and human lymphatic endothelial cells co-culture with cancer cells models are successfully established.5.NRP2 could promote capable of immigration,adhere and canaliculization in hLECs via changing the expression of integrinα_Ⅴβ₃ in hLECs,Sema3F bind NRP2 could induce chemical-repulsion between tumor cell and hLECs,it has the function to inhibit the capable of NRP2 to promot lymphangiogenesis.