| Hepatocellular carcinoma(HCC)is one of the most common malignant digestive system tumors.In addition to the blood metastasis,lymphatic metastasis is an important transfer route which is a complicate regulation process with multiple factors and multiple stages.A series of molecules related to the function、structure and contacted on pathways,such as tumor cells-lymphatic related molecules which observed as a whole,would be an important research direction for the occurrence,development,prediction and clinical evaluation of many tumors including HCC.Nowadays,tumor-induced lymphangiogenesis has become a hotspot in the field of tumor metastasis.More and more evidences suggest that new lymphatic vessels not only exist in the periphery of the tumor,but also appear inside the tumor;not only can be used as a prognostic indicator of tumor lymphatic metastasis,it may also be a therapeutic target to prevent tumor lymphatic metastasis.In recent years,with lymphatic related molecules in lymphatic endothelial cells(LECs)found,such as CNTN1、Prox-1、Podoplanin、LYVE-1、SOX18,etc.,which are direct participants of the VEGF-C/D-VEGFR-3/NRP-2 axis that could induce the proliferation,differentiation,maturing of lymphatic endothelial cells and building or remodeling newborn and original lymphatics structure after being activated.Meanwhile,it can promote the chemotaxis,migration,invasion and metastasis of tumor cells.The previous studies of our research group have screened the differentially expressed genes ANXA7 and VEGF-C from Hca-F and Hca-P with high and low lymphatic metastasis potential,respectively,and revealed that the expressions of these two genes may be closely related to tumor lymphatic metastasis.Therefore,studying the interaction between tumor cells and lymphatic endothelial cells,understanding how VEGF-C/D-VEGFR-3/NRP-2 and lymphatic related molecules form a network to co-participate in the processes of tumor development,especially,in the influences of ANXA7 regulation in tumor cells on the expressions of VEGF-C/D-VEGFR-3/NRP-2 and lymphatic related molecules in lymphatic endothelial cells were also worthy of further investigation,which have important theoretical and clinical significances for the mechanisms of lymphangiogenesis and tumor lymphatic metastasis.Objective: 1.Hca-F/Hca-P hepatocellular carcinoma cells(F/P)and their ANXA7 silenced and overexpressed cells were co-cultured with LEC in vitro,respectively.Meanwhile the expressions of VEGF-C/D-VEGFR-3/NRP-2 and lymphatic related molecules CNTN1、Prox-1、SOX18、Podoplanin、and LYVE-1 in different co-cultured LECs of L-F、L-P、L-FA7DOWN、L-FSHUS;L-PA7UP、L-PNCEV were observed to reveal the effects of F/P and their ANXA7 regulations on the expressions of VEGF-C/D-VEGFR-3/NRP-2 and lymphangiogenesis in co-cultured LECs.2.The expressions of VEGF-C/D-VEGFR-3/NRP-2 and lymphatic related molecules in transplanted tumors of F、P、FA7DOWN、FSHUS、PA7UP、 PNCEV cells and metastatic lymph nodes of L-P 、 L-F 、 L-FA7 DOWN 、FSHUS 、 L-PA7 UP 、L-PNCEV were detected in vivo respectively.Especially,the situations of lymphangiogenesis in above different tissues,the transplanted tumor growth rates and lymph node metastasis rates in six xenografts mice groups of F、P、FA7DOWN、FSHUS、PA7UP、PNCEV tumor cells,and the effects of Hca-F/P cells and their ANXA7 regulation on the expressions of VEGF-C/D-VEGFR-3/NRP-2 and lymphatic related molecules,especially on the formation of lymphatic,were observed in transplanted tumors and metastatic lymph nodes in vivo.Methods: 1.The stable cell lines with ANXA7 silenced and overexpressed Hca-F and Hca-P cells were established and amplified;F/P and stable transfected cells of FA7DOWN、FSHUS、PA7UP、PNCEV by lentivirus were co-cultured with LEC,respectively.2.q RT-PCR,Western Blot and Cytoimmunofluorescence methods were used to detect the expressions of VEGF-C/D—VEGFR-3/NRP-2 and lymphatic related molecules CNTN1、Prox-1、SOX18、 Podoplanin、and LYVE-1 in LEC and different co-cultured LECs of L-F、L-P;L-FA7DOWN、L-FSHUS、L-PA7UP、L-PNCEV at m RNA and protein levels.3.The expressions of VEGF-C/D in supernatants of L-F、L-P、LEC、L-FA7 DOWN,、L-FSHUS、L-PA7UP、L-PNCEV co-cultured lymphatic endothelial cells were detected by ELISA technology.4.The effects of above 6 groups of tumor cells on lymphatic vessel forming ability of LECs were observed by lymphangioplasty.5.Tumor lymphatic metastasis models in mice of stable transfected FA7DOWN、FSHUS、PA7UP、PNCEV cells were established,with eight mice each group;transplanted tumor formations and metastasis to lymph nodes of above six tumor cells in vivo were observed by animal living imaging system,visual observation and HE staining on the days of 7th,14 th,21th and 28 th after injected.On the 28 th day,the mice were executed in the manner of posterior ophthalmic artery blood and the serum,corresponding tumors and lymph nodes were retained.6.At m RNA and protein levels,the expressions of VEGF-C/D—VEGFR-3/NRP-2 and lymphatic related molecules in metastatic lymph nodes of transplanted tumors,normal lymph node and mice serum were detected by q RT-PCR、Western Blot、Cytoimmunofluorescence and ELISA methods.7.The density,distribution,lumen area of lymphatics and expression of lymphatic markers in mouse transplanted tumors and metastatic lymph nodes were labeled by specific lymphatic molecular biomarkers LYVE-1 and Podoplanin,and the data were analyzed by Image J soft.Results: I、The effects of Hca-F/P cells on the expressions of VEGF-C/D—VEGFR-3/NRP-2 and lymphatic related molecules in LECs co-cultured with tumor cells in vitro.1.At m RNA and protein levels,the expressions of ligand VEGF-C/D、receptors VEGFR-3/NRP-2 and lymphatic related molecules SOX18、Podoplanin、LYVE-1 in co-cultured LECs of L-F、L-P were all higher than that in normal LEC,moreover,Podoplanin and LYVE-1 were more obvious,while the expressions of CNTN1 and Prox-1 were all lower in that of co-cultured LECs,and CNTN1 was significantly different.The expression of VEGF-C was slightly lower than that of VEGF-D,while NRP-2 is slightly higher than VEGFR-3 in LEC and co-cultured LECs of L-F and L-P.The expressions of VEGF-C/D,NRP-2/VEGFR-3 and lymphatic related molecules Podoplanin,LYVE-1 and SOX18 in L-F co-cultured cells were all higher than that in L-P co-cultured cells.The expressions of CNTN1 and Prox-1 were lower in L-F co-cultured cells than that in L-P co-cultured cells,and the expression changed amplitude of Podoplanin,LYVE-1 and CNTN1 were significantly in each group.2.The expression of VEGF-C was significantly higher than that of VEGF-D in supernatant of LEC and L-F 、 L-P co-cultured cells.Moreover,the expression differences were the largest in L-F co-cultured cells,and the smallest in LEC.3.The results of lymphangioplasty showed that the number of lymphatics stimulated by being co-cultured with F cells was more than that with P cells.The total length of the lymphatic lumens was greater in co-cultured LECs co-cultured with F cells than that with p-cells and also was larger than of in normal LECs.4.Cytoimmunofluorescence results showed that the expressions of VEGF-C/D were mainly located in cytoplasm;VEGFR-3/NRP-2 were mainly located in cell membrane,with a small amount in cytoplasm;the related molecules CNTN1、Podoplanin and LYVE-1 were mainly located in cytoplasm.Prox-1 and SOX18 were mainly located in nucleus and small in cytoplasm.II、The effects of ANXA7 silenced and overexpressed in F and P cells on the expressions of VEGF-C/D-VEGFR-3/NRP-2 and lymphatic related molecules in co-cultured LECs in vitro.1.The cell lines of FA7DOWN、FSHUS、PA7UP、PNCEV transfected by lentivirus were successfully established and stable amplified with the transfection efficiencies were all more than 90%,and the ANXA7 gene was successfully silenced and overexpressed in F and P cells.2.At m RNA and protein levels,the expressions changes of ligand VEGF-C/D、receptors VEGFR-3/NRP-2 and lymphatic related molecules SOX18、Podoplanin、LYVE-1 in co-culrured LECs of L-FA7 DOWN and L-PA7 UP were all corresponding with the changes of ANXA7 silenced and overexpressed in F and P cells.When ANXA7 was silenced,the expressions of above mentioned molecules were reduced,and they were all increased while ANXA7 was overexpressed.However,the expression of CNTN1 and Prox-1 were opposite to ANXA7 silencing or overexpression.Among them,Podoplanin 、 LYVE-1 and CNTN1 were more significantly affected by ANXA7 regulations.The expression changes of VEGF-C were all larger than VEGF-D in L-FA7 DOWN and L-PA7 UP co-cultured cells,similarly,the expression changed amplitudes of VEGFR-3 were all larger than that of NRP-2.3.The expressions of VEGF-C was significantly higher than VEGF-D in supernatant of L-FA7DOWN、L-FSHUS、L-PA7UP、L-PNCEV co-cultured cells.Moreover,the expression differences were the largest in L-F co-cultured cells,and the smallest in LECs.In addition,VEGF-C/D showed a consistent decreased or elevated trend with the regulation of ANXA7,and VEGF-C changes more obviously.4.The results of lymphangioplasty showed that the number of lymphatics stimulated by being co-cultured with PA7 UP cells was more than that with PNCEV cell,moreover,which was in accordance with the change directions of ANXA7 overexpressed or silenced regulation,namely that the numbers of lymphatics were decreased in L-FA7 DOWN co-cultured cells compared with L-FSHUS group while ANXA7 was silenced, moreover,the total length of the lymphatic lumens was decreased;while it was increased as ANXA7 was overexpressed.III、The expressions of VEGF-C/D—VEGFR-3/NRP-2 and Lymphatic related molecules in transplanted tumor and metastastic lymph nodes of F and P cells in vivo.1.On the 14 th day,the transplanted tumor volume in mice foot was increased after tumor cells were injected;the 21 th day,the swollen lymph nodes in groin could be reached;the 28 th day,the some lymph nodes in the groin,popliteal fossa,armpit,peritoneum,and besides the iliac artery were enlarged.2.HE staining results showed that the transplanted sites of F group were all tumor formed on the 28 th days,the tumor formation rate was 100%,and 6 mice had lymph node metastasis,the metastatic rates was 75%.In the P group,tumors were found in 6 mice,the tumor formation rate was 75%,and only 2 mice had lymph node metastasis,the metastatic rate was 33%.3.q RT-PCR、Western Blot、Cytoimmunofluorescence and ELISA results revealed that at m RNA and protein levels,the expressions of VEGF-C/D、VEGFR-3/NRP-2 and lymphatic related molecules Podoplanin、LYVE-1、SOX18 in metastatic lymph nodes of F、P cells in vivo were all higher than that in normal lymph nodes,moreover,they were higher in F group than in P group,while the expressions of CNTN1 and Prox-1 in metastasizing lymph nodes of F、P cells were all lower than that in normal lymph nodes,moreover,they were lower in F group than in P group,and the expression changed amplitude of Podoplanin,LYVE-1 and CNTN1 were significantly.The expressions of VEGF-C was lower than that of VEGF-D in metastatic lymph nodes,similarly,NRP-2 was higher than VEGFR-3.4.In the serum of xenografts mice,the expressions of VEGF-C/D in F/P groups were all higher than that in normal group,moreover,they were higher in F group than in P group,and VEGF-C was significantly higher than VEGF-D in these three groups.5.Detection of lymphatic specific markers revealed that the number and area of microlymphatics in mice transplanted tumor and metastatic lymph nodes of F cells were significantly higher than that in P cell group,meanwhile,the increase of lymphatic vessels around the tumor was more obvious in F cell group.IV、The effects of ANXA7 silenced and overexpressed in F and P cells on the expressions of VEGF-C/D-VEGFR-3/NRP-2 and lymphatic related molecules in mice transplanted tumors and metastatic lymph nodes in vivo.1.On the 14 th day,the green fluorescence marked in tumor cells could be seen at the site of transplanted tumor in vivo by animal living imaging system which demonstrated the formation of the transplanted tumor;the 21 th day,the green fluorescence could be seen in the groin etc,indicated that tumor lymph node metastasis could be confirmed.2.HE staining results showed that 6 mice were tumor formed in FA7 DOWN group in vivo,the tumor formation rate was 75% and lymph nodes metastasis in 3 mice,the metastatic rates was 50%;8 mice were tumor formed in FSHUS group,the tumor formation rate was 100% and lymph nodes metastasis in 6 mice,the metastasis rates was 75%;8 mice were tumor formed in PA7 UP group,the tumor formation rate was 100% and lymph nodes metastasis in 5 mice,the metastasis rates was 62.5%;6 mice were tumor formed in PNCEV group,the tumor formation rate was 75% and lymph nodes metastasis in 2 mice,the metastasis rates was 33.3%,which were consistent with the animal living imaging results.3.q RT-PCR、Western Blot、Cytoimmunofluorescence and ELISA methods revealed that at m RNA and protein levels,the expressions of VEGF-C/D、VEGFR-3/NRP-2 and lymphatic related molecules Podoplanin、LYVE-1、SOX18 in metastatic lymph nodes of FA7DOWN、FSHUS、PA7UP、PNCEV cells in vivo were all corresponding with the direction of ANXA7 silenced and overexpressed regulation in F and P cells.When ANXA7 was silenced,the expressions of above mentioned molecules were reduced,and they were all increased while ANXA7 was overexpressed.However,the expressions of CNTN1 and Prox-1 were opposite to ANXA7 silencing or overexpression.Among them,Podoplanin,LYVE-1 and CNTN1 were significantly more affected by ANXA7 regulation.The expression of VEGF-C was lower than that of VEGF-D;NRP-2 was higher than VEGFR-3.The reduced or increased amplitude of VEGF-C with the same direction as ANXA7 changes was larger than VEGF-D in transplanted tumors and metastatic lymph nodes of FA7 DOWN and PA7 UP cells,similarly,the same variation amplitude as ANXA7 regulation of VEGFR-3 was larger than NRP-2.4.With the silence and overexpression of ANXA7,VEGF-C/D showed a consistent change with ANXA7 regulation in serum of FA7DOWN、PA7UP、FSHUS、PNCEV xenografts mice.When ANXA7 was silenced,the expressions of VEGF-C/D were reduced,and they were all increased while ANXA7 was overexpressed.The magnitude of expression changes of VEGF-C was significantly higher than that of VEGF-D in supernatant of above four group xenografts mice.5.Detection of lymphatic specific markers revealed that the number and area of microlymphatics in mice transplanted tumors and metastatic lymph nodes of FA7DOWN/PA7 UP cells were significantly higher than that in corresponding control groups of FSHUS、PNCEV、L-FSHUS、L-PNCEV cells in vivo,showing a consistent trend with ANXA7 silenced and overexpressed.When ANXA7 was silenced in F cells,the number and area of microlymphatics in mice transplanted tumors and metastatic lymph nodes of FA7 DOWN cells were all reduced,and they were increased in PA7 UP cell group in vivo while ANXA7 was overexpressed in P cells,moreover,the changes of lymphatic vessels around the tumor were obvious in FA7 DOWN and PA7 UP cell group in vivo.Conclusions: 1.The experiment results in vitro and in vivo consistent indicated that the expressions of VEGF-C/D-VEGFR-3/NRP-2 and lymphatic related molecules Podoplanin、LYVE-1 and SOX18 in lymphatic endothelial cells were positively correlated with the potential of tumor lymphatic metastasis which was positively regulated by ANXA7 regulations.However,the expressions of CNTN1 and Prox-1 were negatively correlated with the potential of tumor lymphatic metastasis which was negatively regulated by ANXA7 regulations.2.The effect of ANXA7 regulation on VEGF-C was greater than that of VEGF-D and that of on VEGFR-3 was greater than on NRP-2.Moreover,Podoplanin、LYVE-1 and CNTN1 were more significantly affected by ANXA7 regulations.VEGF-C、VEGFR-3、Podoplanin、LYVE-1 and CNTN1 were more closely related to lymphatic metastasis of hepatocellular carcinoma,suggesting that these molecules might play a major role in the process of lymphatic metastasis of hepatocellular carcinoma.3、The results of ELISA in vitro and in vivo consistent displayed that VEGF-C/D play a role mainly in the formation of secretory proteins in the process of tumor lymphangiogenesis.Moreover,VEGF-C plays a dominant role.The hepatocellular carcinoma cells of F,P co-cultured with LEC promoted the secretion of VEGF-C/D,especially VEGF-C,and the effect of F cell with high lymphatic metastasis potential is stronger than P cell with low lymphatic metastasis potential.ANXA7 has a positive regulation effect on VEGF-C/D secretion,moreover,the effect on VEGF-C secretion was stronger than that on VEGF-D.4、Lymphangioplasty of LECs in vitro,tumor formation rate and lymphatic metastasis rate,microlymphangiometry in vivo,etc.consistent indicated that the promoting effect of F cells with high lymphatic metastasis potential on the formation and generation of lymphatic was stronger than that of P cells with low lymphatic metastasis potential.ANXA7 regulation in tumor cells is positively correlated with the formation and generation of lymphatic in vitro and in vivo,which plays a positive role in the process of lymphangiogenesis and lymphatic metastasis.5.Based on the results of the study in vitro and in vivo,we can draw a conclusion that in mice liver cancer,ANXA7 may be an important pathway molecule at the upstream of VEGF-C/D-VEGFR-3/NRP-2,which together with the latter regulate the expressions of lymphatic related molecules SOX18,Podoplanin、LYVE-1、CNTN1 and Prox-1 in downstream,especially through the system of specific lymphatic growth ligands,receptors and related molecular such as VEGF-C、VEGFR-3、Podoplanin、 LYVE-1 etc.,playing a catalytic role in the processes of lymphangiogenesis and lymphatic metastasis of hepatocellular carcinoma. |
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