The Effect Of Matrix Metalloprotainases-9 On TNFR Ⅰ Expression And Release Of Hepatocellular Carcinoma Cell

BackgroundsLiver cancer is one of common malignant tumor,accounting for the second malignant tumor in our country,there are nearly 260,000 new cases of primary liver cancer each year,and nearly half happened in our country.The primary liver cancer incidence rate in our country and Southeast Asia is about 5～10 times than Europe and the United States.Male-standardized mortality rate up to 14.52/ 100,000,howere, women standardized mortality rate is 5.61 / 100000.At present,surgery is still thought to be the most important method of treatment.However,because liver cancer is tend to be no symptoms early,when we seek medical treatment in most patients that surgery has lost the opportunity which lead to the overall 5-year survival rate of only 38-47%.In recent years,with the development of medical imaging,clinical biochemistry and technological progress,the rate of early detection of primary liver cancer has been greatly increased and the treatments has taken good progress,too. Along with the development of molecular biology,immunology theory,gene tran.fer technology and pathogenesis of tumor in-depth understanding,the gene therapy research focus gradually from single-gene defects caused by hereditary diseases to multiple gene and steps caused diseases in recent years.Because of liver cancer's high degree of malignancy and poor prognosis that treatment can't significantly prolong the survival time and improve quality of life of patients that treatment of genetically modified organisms has become hot.Tumor cell membrane tumor necrosis factor receptor I(Membrane-bound tumor necrosis factor receptor I,mTNFRI) is one of important death receptors with which TNFα-mediated to kill tumor cells.In vitro and in vivo experiments It has confirmed that mTNFRI involves in mediating programmed cell death(apoptosis), clearing virus infected liver cells and killing tumor cells by combining with tumor necrosis factor(Tumor necrosis factorα,TNFα).However,mTNFRI can be cleaved into the form of sTNFRI by some proteolytic enzymes.The sTNFRI can be combined with TNFαas an antagonist of mTNFRI which exercises an important immunosuppressive effects in vivo.Our group has found that there was a high level of serum sTNFRI in patients with colorectal cancer,which has a positive relationship with the patients' condition,disease course,its recurrence and metastasis.A number of studies aboard confirm this opinon and suggest that sTNFRI can be used as a marker of malignant tumors.Thus stabilizing the mTNFRI is essential for anti-tumor therapy.Matrix metalloproteinases(matrix metallop roteinase,MMPs) are a class of Zn²⁺ with endogenous proteolytic enzymes which are key factors in tumor cell invasion and metastasis.The current researches highlight that high expression and high activity matrixmetalloproteinase(matrix metalloproteinase,MMPs) in almost all tumors. MMPs not only degrade the basement membrane and extracellular matrix of most components,is also involved in the release of a variety of cytokines and / or growth factor and its receptor,such as vascular endothelial growth factor(vascular endothelial growth factor,VEGF),Fas(one of the death receptor family ),which binding to the cell membrane surface as a previous form.In order to achieve tumor invasion and metastasis,MMPs promote tumor cell shedding and destroy basement membrane,matrilysin and angiogenesis.MMP-9 is one of MMPs what has been studied most extensively which is thought to be the most closely related to transfer and invasive in tumor.However,domestic and international fingdings about MMP-9 expression in liver cancer tissue and liver cancer cells have been no uniform conclusion.There are studies shown that MMP-9 in hepatocellular carcinoma was significantly higher than in adjacent tissues,and prompts that may be related to liver cancer prognosis.At the same time,the conflicting view exists:MMP-9 in hepatocellular carcinoma tissues are equal to those in the surrounding non-tumor tissues,with no significant correlation with the clinicopathological features.The research about sTNFRI are so much that there are a number of studies show that patients with malignant tumors who have a high level of body fluids sTNFRI,its sensitivity was even higher than the standard tumor markers.Studies have shown that sTNFRI can even be used to evaluate the malignant.Our group has found high-level sTNFRI exsist in serum and ascites in patients with liver cancer,which suggesting that may be related to immune function in patients with advanced liver cancer.This may be contribute to observe the immune status of liver cancer patients which have a certain significance.Consequently,we propose a new theoretical perspective:mTNFRI falling off to form sTNFRI may be an important mechanism to suppress immune function and become immune escape,in which MMP-9 may play a key role.Therefore,stability of mTNFRI on tumor cells and inhibition of its release may become a new liver cancer treatment targets.Objective1.Organize level:detection the expression of MMP-9 in liver cancer tissues and adjacent tissues,simultaneously we detected serum sTNFRI levels in the corresponding liver cancer patients,in comparison with hepatitis B and the normal control group to confirm whether patients with liver cancer at the same time high expression of MMP-9 and sTNFRI,then to analyze whether there is relevance between the high expression of both;2.Cellular level:Analyze the Change in HepG2 liver tumor cells surface expression of TNFRI before and after using recombinant human MMP-9 interven,and so did sTNFRI in the supernatant.Significance:â‘ Reveal mTNFRI expression and the release of sTNFRI in the tumor cells and its clinical significance;â‘¡MMP-9 takes a key role in clearing mTNFRI falling off,which explores a new target for treatment of liver cancer.Materials and methods1.All the tissues and blood specimens are collected in hepatobiliary surgery department of southern hospital.All cases are confirmed to be hepatocellular carcinoma by postoperative pathology.2.From the organizational level to detect the expression of MMP-9 in hepatocellular carcinoma and adjacent tissues,while serum sTNFRI levels in the corresponding liver cancer patients,hepatitis B and the normal control group to confirm whether liver cancer patients have high expression of MMP-9 and sTNFRI simultaneously, then to analyze whether the high expression of both existence relevance.3.From cell level to further verify the correlation between MMP-9 and the sTNFRI1)Cultivating hepatoma cell line HepG2 to extract cell RNA and protein for RT-PCR and Western experiments respectively,then verifing existence of the high expression of TNFRI in HepG2 from the gene level and protein level.2)Effect of MMP-9 in expression of TNFRI on hepatocellular carcinoma HepG2 cell surface:Afer transferred in 6cm diameter petri dishes,HepG2 were cultured in incubator 37℃,5%CO₂ for 12h.Then changed serum-free medium.after 24h,added RPMI-1640 including active MMP-9,total volume 1000 ul.divided into groups: â‘ MMP-9 0ng/mL,â‘¡MMP-9 154ng/mL,â‘¢MMP-9 385ng/mL,â‘£MMP-9 770ng/mL,⑤MMP-9 1155ng/mL 3h,or MMP-9 770ng/mL 0,1.5,3,6 and 9h.The expression of TNFRI were detected by Western Blotting.All supernatant are stored in -20℃, preparing for the next step.3)Expression of sTNFRI in supernatant of cell culture,stored supernatant were placed in room temperature,after centrifuging at 500×g for 5min,quantitate sTNFRI using ELISA kit.4.Statistical methodsExperimental data expressed as mean±SD,One-way ANOVA,T-Test,LSD and Linear correlation analysis were used to estimate the item differences among the groups using SPSS13.0 for Windows.Results1.Expression of MMP-9 is higher in the liver cancer tissues and it is positively related to portal vein thromb and tumor invasion.But there has no significant difference of TNFRImRNA between liver cancer tissues and the adjacent liver tissues.2.sTNFRI level was significantly higher in liver cancer patients than that of hepatitis B group and normal control group.But there is no significant correlation between the corresponding cancer tissue MMP-9 and sTNFRI.This might results from the multipile resources of sTNFRI and requires further validation from the cellular level.3.The effect of different concentrations of MMP-9 on TNFRI expression.HepG2 incubated 3h was added different concentrations of MMP-9,after 3h,each group showed different quantity expression.The group MMP-9 0ng/mL shows similar expression with group MMP-9 154ng/mL but higher than MMP-9 385ng/mL and group MMP-9 770ng/mL,group MMP-9 385ng/mL shows similar expression with group MMP-9 770ng/mL,which are higher than group MMP-9 1155ng/mL.The results showed MMP-9 had a down regulation effect on the expression of TNFRI in cell membrance of HepG2 and was correlated with dose.4.The effect of MMP-9 on expression of TNFRI at different time.Fixed MMP-9 concentration at 770ng/mL,the differential quantity expression were observed at each activating point,group Oh is similar with group 1.5h but both are higher than group 3h.Group 3h is higher than group 6h and group 9h,.Datas from aboves showed the down regulation effect of MMP-9 on the expression of TNFRI in cell membrance of HepG2 was correlated with activating time.5.The effect of different concentrations of MMP-9 on expression of sTNFRI in cultured HepG2 supematant at 3h.The different expression of sTNFRI were obtained visived in different concentrations quantitated by ELISA at 0ng/mL,48.36pg/mL,at 154ng/mL,52.33pg/mL,at 385ng/mL,84.17 pg/mL,at 770ng/mL,94.45 pg/mL and at 1155ng/mL,142.40ng/mL.These datas showed MMP-9 could promote TNFRI shed from cell surface of HepG2 cell which was correlated with dose.6.Change of sTNFRI in cultured cell supernatant at different time course after adding MMP-9 770ng/mL.The expressions of sTNFRI were different in each group quantitated by ELISA,at Oh:48.38pg/mL;at 1.5h 55.00pg/mL;at 3h 79.85pg/mL;at 6h 125.64pg/mL;at 9h 128.91pg/mL.These datas showed MMP-9 could promote TNFRI shed from cell surface of HepG2 which was correlated with activating time.Conclusion1.The gene and protein Expression of MMP-9 in liver cancer tissue were higher than adjacent group,and it is positively related to portal vein thromb and tumor invasion. But there has no significant difference of TNFRImRNA between liver cancer tissues and the adjacent liver tissues.2.sTNFRI level was significantly higher in liver cancer patients than that of hepatitis B group and normal control group.But there is no significant correlation between the corresponding cancer tissue MMP-9 and sTNFRI.This might results from the multipile resources of sTNFRI and requires further validation from the cellular level.3.MMP-9 had a promotion effect on the process of sTNFRI origined from TNFRI shedding from the HepG2 cell surface,the efficiency depends on treatment duration and dosages.MMP-9 had also a down regulation effect on the expression of TNFRI in cell membrance of HepG2,the effect was probable correlated with invasion and metastasis of liver cancer.