The Effect And Mechanism Of Tumor Necrosis Factor Alpha On Liver Tumorigenesis

?Background and Object?Primary liver cancer is one of the most malignant tumor.The tumor incidence is high,which is the sixth in the world,and the tumor types are various,which include hepatocellular carcinoma(HCC),cholangiocellular carcinoma(CC),and mixed carcinoma.The clinical data show that different type of liver cancer has different clinical outcome.For example,the malignant degree of CC or mixed carcinoma is far higher than that of HCC,and the prognosis of the same liver cancer,but different pathological classification,is also presented variously.These clinical feature is closely related to tumor heterogeneity,and the research about tumor heterogeneity mechanism has become an important focus in recent years.At present,the question about liver cancer origin is still controversial.More and more studies have shown that chronic liver injury is one of the important pathological process leading to liver cancer.Virus infection,high fat diet,excessive drinking,and cholestasis can cause chronic liver injury.This process not only has a large number of cells with necrosis or apoptosis,but also has inflammation throughout.Hepatic Progenitor cells(HPCs)also tend to be activated and participated in repair the chronic liver injury.However,continued liver damage beyond repair leads to liver cancer eventually,how inflammation is involved in HPCs differentiating,and participating in liver cancer heterogeneity,which is still unclear.As an inflammation-related disease,inflammation is accompanied from chronic liver damage to liver cancer.The effect of tumor necrosis factor alpha(TNF-?)has always attracted researchers' attention.TNF-? has been confirmed to promote liver cancer growth,invasion and metastasis,and also be related to clinical prognosis of liver cancer patients.However,the studies about the effect of TNF-? on hepatocarcinogenesis are only limited to a certain stage,the molecular mechanism of which also has a certain one-sidedness.HPCs is also thought to play an important role in liver injury and repair,as well as liver tumorigenesis,and the activated HPCs can not only differentiate into adult hepatocytes and bile duct cells to repair damaged tissue,but also differentiate into liver cancer abnormally.Many studies have shown that chronic inflammation in the process of liver damage influences HPCs activation as well as differentiation direction.Therefore,to nvestigate the effect of TNF-? on HPCs activation and differentiation not only helps us to understand the nature and heterogeneity of liver cancer induced by inflammation,but also provides theoretical guidance for the further intervention.In our research,we establish rat liver cancer model by DEN,and use different doses of TNFR2-Fc fusion protein variant(TNFR2-Fc V)to change TNF-? concentration in the process of liver cancer,as well as Tnf?-/-rats.According to these rat models,we will observe rat liver damage,tumor incidence and liver cancer types,and further analyze the effect of TNF-? on HPCs activation,differentiation,and explore the specific molecular mechanism of which.The aim of our study is in order to clear the nature of liver cancer as well as liver cancer types induced by TNF-?.Part ? The effect of TNF-? on DEN-induced rats' liver tumorigenesisIn order to observe the effect of different doses of TNF-? inhibitor on liver tumorigenesis,we establish DEN induced-rat liver cancer model firstly,and use TNFR2-Fc V to influence TNF-? concentration.Then,we observe tumor incidence and overall survival time of rats,as well as the liver damage index in serum.The results show that 8 mg/kg TNFR2-Fc V treatment can obviously reduce TNF-? concentration in DEN induced-rat,which has no difference with normal rat,while low dose(1 mg/kg)of TNFR2-Fc V also decreases TNF-? concentration significantly,but it is still higher than that in normal rat serum;high dose of TNFR2-Fc V can significantly inhibits DEN induced-liver cancer,while low dose of TNFR2-Fc V has no effect on the tumor incidence of rat;high dose of TNFR2-Fc V reduces liver function index in serum and shortens the lifetime,while low dose of TNFR2-Fc V improves the rat liver function effectively and prolongs the survival period.The results indicate that different doses of TNFR2-Fc V treatment in DEN induced-rat liver cancer model has different effect on TNF-? concentration,as well as tumor incidence,liver damage and survival time.Part ? The effect and mechanism of TNF-? on HPCs activation during liver tumorigenesisIn order to study the influence of high dose of TNF-? inhibitor on HPCs activation in the process of liver cancer,we take out the rat tissue samples treated with high dose of TNFR2-Fc V,and used immunohistochemical and immunofluorescence staining to detect the effect of TNF-? on HPCs activation in this process.The unmarked cell analyzer RTCA),Real-time PCR,and Western-blot are used to detect the influence of TNF-? on HPCs proliferation as well as molecular mechanism;Furthermore,the clinical liver cancer tissue samples are detected by immunohistochemical staining to confirm the key molecular expression of TNF-? affecting HPCs activation.The results show that high dose of TNFR2-Fc V inhibits liver repair in the process of DEN induced-liver cancer,which is companied with inhibition of HPCs activation and proliferation.The experiment in vitro confirms that TNF-? promotes HPCs activation and proliferation through the combination with TNFR2 on the surface of HPCs firstly,and further activating Stat3 signaling pathways.In the clinical tissue adjacent to liver cancer,the expression of TNF-? and HPCs activation index show a positive correlation.The results indicate that the inhibition of TNF-? interferes liver repair in the process of DEN induced-rat liver cancer,which may be related with inhibition of HPCs activation and proliferation mediated by TNFR2/Stat3 signaling pathway,thus high dose of TNFR2-Fc V not only hinders the process of liver cancer,but also causes liver failure and shorter survival.Part ? The effect and mechanism of TNF-? on HPCs differentiation to liver cancer heterogeneityIn order to study the effect and mechanism of low doses TNF-? inhibitor on heterogeneity of liver cancer,we take out the rat tissue samples treated with low dose of TNFR2-Fc V,and used immunohistochemical and immunofluorescence staining to detect the effect of TNF-? on DEN induced-liver cancer type and the change of key signaling pathways.Immunofluorescence staining,Real-time PCR,and Western-blot are used to detect the influence of TNF-? on HPCs differentiation as well as molecular mechanism;finally,the clinical liver cancer tissue samples are detected by immunohistochemical staining to confirm the key molecular expression of TNF-? affecting HPCs differentiation.The results show that low dose of TNFR2-Fc V can reduce HCC/CC structure proportion in DEN induced-liver cancer,TNF-? inducing HPCs to the bile duct cell differentiation is mainly by activating NF-?B signaling pathway to increase Jagged 1 expression in mesenchymal cells,but not through direct effect;In the primary clinical liver cancer tissue,TNF-? expression has a correlation with tumor recurrence type.The results indicate that TNF-? increasing Notch signal pathway to induce bile duct cell differentiation in HPCs is through mesenchymal cells indirectly in the process of liver cancer,if TNF-? is inhibited by low dose of TNFR2-Fc V,HCC/CC proportion in liver cancer is reduced,and rats' urvival time is prolonged,although the tumor incidence has no change.