NK4 Affect Angiogenesis And Invasion Of Cholangiocarcinoma Through HIF-1?/VEGF Pathway

Background:Cholangiocarcinoma(CCA)is relatively rare,accounting for about 3%of all digestive tumors,is currently the second largest primary hepatobiliary malignant tumor after hepatocellular carcinoma(HCC).The incidence of CCA is increasing and the five-year survival rate has not exceeded 10%.The prognosis of CCA was poor,with an average lifetime of less than 24 months.Hidden onset,difficult early diagnosis,non-sensitive to chemotherapy and radiation therapy are possible reasons of poor prognosis.Surgical resection is currently the most effective treatment.However,due to hidden early symptoms,signs,diagnosis methods,lack of effective diagnostic methods,and specific bile duct location,CCA could easily invade neighboring tissues and blood vessels duo to its invasive growth characteristics,resulting in less than 30%CCA patients acquire radical surgery opportunity.Therefore,further studies are needed to seek effective therapy methods for CCA that improving the curative effect and prognosis of CCA.Hepatocyte growth factor(HGF),also known as the discrete factor,shows two different effects in different condition.In normal physiological condition,HGF promotes cell growth and regeneration,which is beneficial for us.While in the tumor tissues,or in other pathological physiology,HGF might promote tumor cell hyperplasia and excessive formation of capillaries,leading to tumor invasion and distant metastasis.c-Met,as a receptor of HGF,play an important role in tumor pathogenesis.Many studies have found that HGF/c-Met pathway is involved in tumor occurrence,invasion and metastasis.NK4 is the first proved antagonist of HGF/c-Met signaling pathway.Its molecular structure is similar to the alpha chain of HGF that NK4 could combine with c-Met,completely competitive inhibiting the combination of HGF and Met,resulting in the failure of c-Met phosphorylation,thus affecting HGF/c-Met signal transduction,finally suppressing cell proliferation and invasion.In addition,NK4 can inhibit vascular endothelial growth factor(VEGF)expression that reducing the production of tissue capillaries.Hypoxia inducing factor 1 alpha(HIF-1?)is an important transcription factor regulation of VEGF through activate endothelial cell surface receptor tyrosine kinase,promoting endothelial cell proliferation,migration and interstitial protein hydrolysis,etc.,participating in the formation of blood vessels.Objective:This study explore the role of NK4 in angiogenesis and invasion of CCA by regulating HIF-1?/VEGF pathway in vitro and vivo.Methods:1.Stable transfection CCA HuCC-T1 lines with NK4(Hu-NK4)and control plasmid(Hu-Em)were treated with HGF exposure to hypoxia and normal oxygen condition.Respectively,MTT,transwell test and flow cytometry analysis were used to analyse cell proliferation,invasion,and apoptosis.2.Stable transfection CCA HuCC-T1/EM and HuCC-Tl/NK4 cells were inoculatedin subcutaneous of nude mice model.The size and weight of tumors were measured and CD31 positive microvascular formation was analyzed immunohistochemically.Realtime-PCR and Western blotting analysis were used to detect the expression of HIF-1?,VEGF and CD31.Results:1.In the condition of hypoxia and normoxia,NK4 overexpression significantly inhibits the proliferation of HuCC-T1 cells.Also,hypoxia significantly enhances the inhibition of NK4 for HuCC-T1 cells.2.Similarly,in the condition of hypoxia and normoxia,NK4 overexpression significantly promoted the apoptosis of HuCC-T1 cells.Hypoxia significantly enhances the apoptosis-promoting role of NK4 for HuCC-T1 cells.3.Hypoxia increases the ability of cell invasion,however,over-expression of NK4 inhibit the invasion effects induced by hypoxia.4.Hypoxia increased the expression of HIF-1?,CD31 and VEGF.However,over-expression of NK4 inhibited this effect and thus reduced angiogenesis.5.In HuCC-T1 mice,NK4 transfection inhibited the growth of the tumor.6.In HuCC-T1 mice,NK4 transfection reduced the expression of HIF-1?,CD31 and VEGF expression,thus inhibiting the production of tumor blood vessels.Conclusions:1.NK4,as HGF antagonist,significantly inhibited the proliferation and invasion of cholangiocarcinoma cell lines,and induced apoptosis in vitro.2.NK4 supressed the expression of HIF1a,VEGF,and CD31,suggesting that NK4 inhibited vascularization of CCA cells induced by hypoxia by regulating HIF1a/VEGF pathway.3.In CCA mice model,NK4 inhibited the expression of HIF-1a,VEGF and CD31 in tumor tissues,suggesting NK4 inhibited the growth of tumor and blood vessels formation in vivo.