Annxein A7 Regulate VEGF-C/R3 Signal Axis Increases The Intratumoral LMVD To Participate The Lymphatic Metastasis Of Hepatocellular Carcinoma

Background: Metastasis is one of the important factors leading to poor prognosis of primary hepatocellular carcinoma(HCC).Although the blood metastasis accounted for 64% in patients with metastases,but 33.8% patients had occurred in extrahepatic lymph node metastasis(LNM)before hematogenous metastasis.Therefore,it is of great practical significance to study the mechanism of lymphatic metastasis of HCC and explore effective methods of diagnosis and treatment.Annexin A7(Anxa7)is a calcium dependent phospholipid binding protein,which has been reported to play an important role in the lymphatic metastasis of HCC.The expression of Anxa7 in mouse hepatoma cell lines with high lymphatic metastasis rates was higher than the hepatoma cell lines with low lymphatic metastasis rates.suggesting that Anxa7 may promote lymphatic metastasis of HCC.But there is another view that Anxa7 has inhibitory effect on hepatoma lymphatic metastasis,researcher found that when the expression of Anxa7 was up-regulated in hepatoma cell line Hca-P the lymph node metastasis rate were reduce and down-regulated the expression of Anxa7 were increase the lymph node metastasis rate in the animal model.the results show that Anxa7 inhibited the lymphatic metastasis.At present,the specific role of Anxa7 in HCC was still controversial, and the specific mechanism was not clear.In the study of lymphatic metastasis,VEGF-C/D/R3 signaling axis was the most important lymphangiogenesis pathway,this pathway can promote lymphatic metastasis in a variety of cancers including HCC,VEGF-C/D secreted by Tumor cells and tumor associated stromal cells combined with VEGFR-3 on lymphatic endothelial cells,then activate the downstream signaling pathways and regulating lymphatic endothelial cell proliferation,migration,and lead to the occurrence of LNM.We predicted that Anxa7 may be involved in the regulation of VEGF-C/D/R3 pathway in hepatocellular carcinoma.Objective: 1.To investigate the role of Anxa7 and VEGF-C/D in the development and lymphatic metastasis of HCC;2.To explore the relationship between the expression of Anxa7 with VEGF-C/D/R3 and the impact on lymphangiogenesis.Method: 1.20 cases of HCC fresh sample was collected in the Liaoning cancer hospital with tumor tissue and paired normal liver tissue,the real time PCR and Western blot will used to detect the expression of Anxa7,VEGF-C,VEGF-D in the tumor tissue and normal liver tissue both the gene and protein level;2.Immunohistochemical was used to detect the expression of Anxa7,VEGF-C and VEGF-D in HCC of 160 cases,immunohistochemistry D2-40 staining to count the Lymphatic microvessel density(LMVD).3.We select the Human liver cell line THLE-2?Human HCC cell line Hep G2 with normal VEGF-C/D expression and the Human HCC cell line SK-Hep-1 with low VEGF-C/D expression,and divided the cell into treated group and untreated group,the treated group up-regulate the Anxa7 expression by add human Anxa7 recombinant protein,and the untreated group don,t add human Anxa7 recombinant protein.Then detected the expression of Anxa7,VEGF-C/D by real time PCR and Western blot,the secretion of VEGF-C/D in supernatant was detected by ELISA.4.The liver cell line and HCC cell line will used Co-culture with HDLEC cell and uesed tube formation experiment to explore the impact of Anxa7 expression on lymphangiogenesis,real time PCR and Western blot used to detect the expression of VEGFR-3 in HDLEC.Results: 1.We found that the expression of Anxa7 and VEGF-C in tumor tissues was significantly higher than normal tissues(P=0.006,0.027)in the m RNA level,there was no significant difference about VEGF-D(P=0.167);At the protein level,Anxa7 and VEGF-C expression in tumor tissue was also greater than the expression in normal tissues(P=0.047,0.04),the expression of VEGF-D between tumor tissue and normal tissue was no difference(P=0.64).Compare the LNM group and the un-LNM group,the expression of Anxa7 and VEGF-C in the LNM group was higher(P=0.01,0.01)than the un-LNM group in m RNA level,while there was no significant difference in the the m RNA level about the VEGF-D expression(P=0.186);at the protein level,the expression of Anxa7 and VEGF-C in LNM group was higher than the un-LNM group(P=0.02,0.01),while the expression of VEGF-D protein in the two groups have no significant difference(P=0.56).2.Compared the LMVD,the results showed that the LMVD of intratumoral tissue was significantly higher than that in peritumoral tissue(P=0.01);Comparison of LNM group and un-LNM group,the intratumoral LMVD in LNM group was significant higher than the un-LNM group(P=0.01)and there was no significant difference between LNM group and un-LNM group about peritumoral LMVD(P=0.26).We test the relationship between VEGF-C?VEGF-D expression and LMVD,and found that there is a correlation between VEGF-C m RNA expression and the intratumoral LMVD(P=0.00),but was not correlated with VEGF-D m RNA(P=0.348).In the protein level,the VEGF-C exprssion was correlated with the intratumoral(P=0.04),and the expression of VEGF-D was not correlated with the intratumoral LMVD(P=0.85).3.In order to investigate the relationship between Anxa7 and VEGF-C/D/R3 signaling axis and intratumoral LMVD,we analyzed the correlation between the expression of Anxa7 and VEGF-C/D and intratumoral LMVD,found that there is a relationship between Anxa7 m RNA expression and VEGF-C m RNA expression and intratumoral LMVD(P=0.00,0.00),and not correlated with VEGF-D m RNA expression(P=0.638);Anxa7 protein was correlated with the VEGF-C protein expression and intratumoral LMVD(P=0.00,0.00),and while there was no significant correlation between Anxa7 protein and VEGF-D ptotein(P=0.92).4.The results of immunohistochemistry showed that Anxa7,VEGF-C and VEGF-D was expressed in tumor cells and normal liver cell cytoplasm.And the expression of Anxa7,VEGF-C in tumor tissue was stronger than the liver tissue,and the expression of VEGF-D between tumor tissue and liver tissue was not different;And the expression of Anxa7 and VEGF-C in LNM group was higher than in un-LNM group,the expression of VEGF-D in the two groups was not difference.Spearman correlation test showed that the expression of Anxa7 and VEGF-C was negatively correlated to the differentiation of HCC,and was positively correlated with tumor size,TNM stage,LNM.The survival analysis showed that high expression of Anxa7 and VEGF-C means lower 5 year survival rate of patients with HCC,the expression of Anxa7 and VEGF-C was independent prognostic factor.5.The expression of Anxa7 in THLE-2?Hep G2 and SK-Hep-1 was up-regulated by human recombinant Anxa7 protein.And we detected the expression and secretion of VEGF-C/D in THLE-2,Hep G2 and SK-Hep-1 cells and the expression of VEGFR-3 in co-cultured HDLEC and HUVEC cells line.The results showed that the expression of VEGF-C in THLE-2?Hep G2 and SK-Hep-1 was increase,the secretion of VEGF-C in supernatant was increased too.but the expression and secretion of VEGF-D was not change.In HDLEC cells,the expression of VEGFR-3 increased at both gene level and protein level.6.Compare the tube formation between treated group and untreated group,found that the tube formation of HDLEC in treated group was higher than untreated group,but the tube number of HUVEC cell between treated group and untreated group was not change.7.There was no interaction between Anxa7 and VEGF-C.Conclusion:1.Anxa7 and VEGF-C can promote the occurrence and development of HCC and lymphatic metastasis,which is an independent factor affecting the prognosis of HCC patients;2.Anxa7 could increase the expression and secretion of VEGF-C to promote the expression of VEGFR-3 in lymphatic endothelial cells,and then increase the formation of intratumoral lymphatic vessels to promote lymphatic metastasis of HCC.