The Role And Mechanism Of Chemokine CCL20-induced Epithelial-Mesenchymal Transition In Invasion And Metastasis Of Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is the sixth most common cancer and the third cause of cancer-associated death which presents gradual upward trend in recent years.Most radical resection of liver cancer can be obtained with the development of science and technology and surgical techniques,but the survival rate remains low in the overall 5 years.The most important factor is that HCC extremely had the ability of invasion and metastasis.Tumor recurrence and distant metastasis are very prone to take place in 5 years after radical resection of hepatocellular carcinoma.So,HCC recurrence and metastasis after operation are our clinical problems.Tumor microenvironment(TME)is a special circumstance formed by the progress of tumor cell growth,which is caused by the interaction between tumor cells and extracellular matrix and TME included tumor cells,a variety of mesenchymal and inflammatory cells,soluble cytokines,and extracellular matrix and so on.It has played an important role in the process of tumor development and metastasis.Epithelial-mesenchymal transition(EMT)refers to the biological process that epithelial cells transfer into mesenchymal phenotype cells and have their characteristics.Currently,most scholars regarded EMT as an important way of tumor invasion and metastasis.Chemokines,which are components of the tumor microenvironment,are also involved in the tumorigenesis and development of various tumors.Some papers already proved that chemokines could induce tumor cells that promote the invasion and metastasis of tumor cells in EMT way.CCL20,which is a hot topic in recent years,has been proven to play a very important role in tumor invasion and metastasis.Meanwhile,it also plays similar important role in HCC.Scholars have found that CCL20 and CXCL8 can synergize and then promote the invasion and metastasis of colorectal tumor cells in EMT-like way.On the other hand,our previous study showed that the co-culture between the conditioned medium of tumor-associated fibroblasts in the liver cancer microenvironment and liver cancer cells could significantly enhance the ability of invasion and metastasis of HCC cells.Presumably,it mainly depended on that the tumor-associated fibroblasts affect the biological behavior of liver cancer cells via autocrine way.Actually,the specific mechanism is not very clear.Studies have shown that tumor-associated fibroblasts could secrete a variety of cytokines including CCL20.Apoptosis which is also called programmed cell death(PCD)means that cells could die physiologically according to their intrinsic program on some certain physiological and pathological conditions.The inhibition of apoptosis was taken as the prerequisites for the survival and metastasis of tumor cells.It had been proven that chemokines and apoptosis play essential roles in the progression of HCC.Vascular endothelial growth factor-related chemokine 1(VCC-1),a kind of chemokine,was proved to relate with the HepG2 proliferation and inhibition of HepG2 cells' apoptosis.Based on the above background,we hypothesized that chemokine CCL20 in HCC microenvironment might promote HCC cells' invasion and metastasis in an EMT way and inhibit the apoptosis of HCC cells in the meantime.So far,there was no similar report and then in order to verify our hypothesis,the study mainly included the following aspects:(1)The expression of CCL20 in normal liver cells(LO2)and different HCC cell lines were detected,including HepG2,MHCC-97 H,Hep3B,SMMC-7721 and Huh7,and then one hepatocellular carcinoma cell line which showed high-expressed CCL20 was selected;(2)We observed that the difference between CCL20 and siRNA-CCL20 on the EMT-like changes and the biological properties changes of liver cancer cells in vitro.We used immunofluorescence to detect the expression differences of EMT-associated protein(E-cadherin,vimentin)in liver cancer cells;(3)Through MTT,Transwell and woundhealing assays,we tested the changes of invasion and migration of liver cancer cells in CCL20 group and siRNA-CCL20 group;(4)We detected the expression difference of EMT-associated protein and signaling pathways associated protein by Western blot;(5)By immunohistochemistry,CCL20 and EMT-related protein were detected in liver cancer samples and adjacent tissues;(6)In the meantime,clinicopathological data and follow-up data of HCC patients were also collected.In order to obtain the correlation between CCL20 and clinical outcomes,data analysis and statistical analysis were performed.The study is divided into three parts.Part I CCL20 promotes the ability of invasion and metastasis in liver cancer cells by inducing EMT-like changes.Objective: To clarify whether CCL20 can induce EMT-like changes of liver cancer cells and promote the invasion and metastasis.Methods: First,we detected the CCL20 expression in normal liver cells(LO2)and different liver cancer cell lines(HepG2,MHCC-97 H,Hep3B,SMMC-7721 and Huh7)by Western blot and chosed the highest CCL20 expression HCC cell lines to following experiment;Next,through MTT,Transwell,we tested the invasion and migration changes in hepatocellular carcinoma cells which were induced by CCL20.After co-culture with CCL20 and Hep3 B cells,the morphology changes of Hep3 B cells can be observed.Results: Western blot analysis showed that the expression of CCL20 in liver cancer cells was significantly higher than that in normal liver cells and the expression in Hep3 B performed the highest.MTT revealed that CCL20 could improve the proliferation rate of Hep3 B cells in a concentration and time-dependent manner and Transwell also showed that CCL20 could promote the migration of the Hep3 B cells in a concentration-dependent manner.After the treatment with CCL20 for 24 h and 48 h,the results showed that CCL20 could induce the morphology changes of Hep3 B cells such as spindle-shape mesenchymal morphology and a loose association and the morphology changes which were more obvious with the increase of CCL20 concentrations.Conclusions: CCL20 could promote cancer cell invasion and metastasis by inducing EMT-like changes.Part II Down-regulated CCL20 could inhibit the proliferation,invasion and metastasis,and apoptosis of liver cancer cells.Objective: To clarify whether siRNA-CCL20 could inhibit the proliferation,invasion and metastasis,and apoptosis of liver cancer cellsMethods: First,through MTT,Transwell and wound-healing assays,we tested the invasion and migration changes in hepatocellular carcinoma cells which were induced by siRNA-CCL20;Then,the difference of EMT markers was tested by immunofluorescence;Next,Western blot was carried out for the detection of EMT markers and related signaling pathways proteins;Finally,the apoptosis effect of CCL20 on Hep3 B cells was tested by flow cytometry and Western blot was carried out for the detection of apoptosis-related proteins.Results: MTT,Transwell and wound-healing assays showed that siRNA-CCL20 could inhibit the proliferation rate and invasion and metastasis of Hep3 B cells.After co-culture with siRNA-CCL20 and Hep3 B cells for the treatment for 48 h,we could see that the expression of E-cadherin was up-regulated while the expression of Vimentin,?-catenin and pAkt were down-regulated in the siRNA-CCL20 group and no changes for the pERK.Through Western blot and flow cytometry,we could see that the expression of cleaved caspase-3 was up-regulated while the expression of Bcl-2 was down-regulated in the siRNA-CCL20 group which was similar to the results of flow cytometry.Conclusions: Down-regulated CCL20 could inhibit the proliferation,invasion and metastasis,and apoptosis of liver cancer cells and the possible signal pathway was PI3K/AKT.Part III The correlation between CCL20 expression,EMT-related protein expression and prognosis.Objective: To detect the expression of CCL20 and EMT-related protein in HCC and determine their relationship between the clinicopathological features and prognosis of HCC patients.Methods: First,we used immunohistochemistry test to identify the expression of CCL20,E-cadherin and Vimentin in the liver cancer tissue and para-carcinoma tissue and then according to the staining degree of CCL20,we divided 102 patients into high and low expression group and then analyzed their date with EMT-related protein and clinicopathological data.Next,we used multivariate regression analysis to predict poor prognosis of liver cancer.Last,we used Kaplan-Meier method and log-rank test to compare the overall survival rate and disease-free survival rate in two groups.Results: CCL20 expression mainly located in liver cancer cell cytoplasm and about 77.45%(79/102)CCL20 were expressed in HCC,but only 20.59%(21/102)CCL20 were expressed in adjacent liver tissue.We also found that CCL20 expression was accompanied with decreased expression of E-cadherin and increased expression of Vimentin.102 patients were divided into high CCL20 expression group(64/102)and low expression group(38/102)and high CCL20 expression was negatively correlated with the expression of E-cadherin(13/64,20.31%,P<0.001)and positively correlated with the expression of Vimentin(60/64,93.75%,P<0.001).Through further study of the patients' pathology and clinical data,we found that the CCL20 expression was significantly correlated with tumor size(P=0.001),vascular invasion(P=0.025),distant metastasis(P=0.000)and intra-hepatic metastasis(P=0.002).Using multivariate Cox regression analysis,we could find that CCL20 expression,vascular invasion,intrahepatic metastasis and distant metastasis could significantly affect overall survival and tumor-free survival in HCC patients.Compared with low CCL20 expression group,high CCL20 expression group showed significantly decreased overall survival(P=0.011)and disease-free survival(P=0.008).Conclusions: CCL20 expressed in HCC and correlated with the expression of EMT-related proteins and poor prognosis in hepatocellular carcinoma patients after operation,which suggested that CCL20 might become a new marker in clinical therapy and prognostic prediction of HCC.Summary CCL20 expressed in liver cancer cells and could enhance the ability of proliferation,invasion and metastasis of liver cancer cells by inducing tumor cells into EMT-like changes and inhibition of apoptosis.The possible mechanism may be the regulation of PI3K/Akt signaling pathway.Clinical study demonstrated that high CCL20 expression in hepatocellular carcinoma tissues was related with EMT-related protein.There was obvious correlation between the expression of CCL20 and clinicopathologic data,overall survival and disease-free survival.Therefore,we could conclude that CCL20 might become a potential prognostic indicator to guide individual treatment of liver cancer patients.