The Expression And Role Of MFGE8 In Hepatitis-Liver Cirrhosis-Hepatocellular Carcinoma Progression

In the process of hepatitis-liver cirrhosis-hepatocellular carcinoma progression,cytokines are secreted by necrosis and apoptosis of hepatocytes through autocrine and paracrine ways.Various insult factors including hepatitis virus,alcohol,nonalcoholic fatty liver disease and hepatotoxicant can trigger the activation of hepatic stellate cells and kupffer cells and thus induce the chronic inflammation.Hepatic fibrosis is reversible once the etiological agent is removed.With the reduced number of activated hepatic stellate cells by the induction of apoptosis,collagen deposition is decreased and liver architecture and function are recovered.If the chronic inflammation is prolonged,it would eventually develop into liver cirrhosis or hepatocellular carcinoma.Cytokine,a kind of soluble protein derived from various cells,is stimulated by immunogen and secreted in paracrine,autocrine or endocrine ways.Cytokines play a pivotal role in regulating cell proliferation,differentiation,migration,apoptosis and immune response,and consist of a complex network to maintain various physiological functions.MFGE8 is a secretary glycoprotein,widely participated in the interaction of various cells.MFGE8 plays an important role in inflammatory regulation,tissue fibrosis as well as cancer development.This study was aimed at screening the vital cytokine in hepatitis-liver cirrhosishepatocellular carcinoma progression.The study was consisted of the following three parts.Part 1 Screen of the pivotal cytokine involved in hepatitis-liver cirrhosishepatocellular carcinoma progression by cytokines arrays analysis.Cytokines arrays were customized to analyze the serum of the C3 H mice of normal group,CCl4-induced and DEN/PB-induced group.Four potential cytokines in hepatitis-liver cirrhosis-hepatocellular carcinoma progression were selected for further identification.Real-time PCR analysis indicated that TGF-β was elevated in CCl4-induced group,with 1.7-folds in CCl4 2w group(p < 0.01),while chrodin showed no obvious changes in both CCl4-induced and DEN-induced groups.In DEN 2m,DEN 5m and DEN 8m groups,the expression of prostasin was increased by 2.7-folds,5.4-folds and 2.5-folds,respectively(p < 0.01).Meanwhile,the expression of MFGE8 was significantly increased in CCl4-induced groups by 3.8-folds,3.3-folds and 3.4-folds,respectively(p < 0.001),and gradually increased in DEN-induced group by 1.1-folds,1.6-folds and 2.3-folds,respectively.The results indicated a potential involvement of MFGE8 in hepatitis-liver cirrhosishepatocellular carcinoma progression.Part 2 Expression of MFGE8 in acute and chronic CCl4-induced mouse models.To confirm the expression of MFGE8,we constructed the acute and chronic CCl4-induced mouse models.Immunohistochemical staining indicated the increased expression of MFGE8 in the cytoplasm of hepatocytes in acute and chronic models.Serum MFGE8 expression was assessed by ELISA,and results showed that MFGE8 was significantly increased in CCl4-induced acute injury(p < 0.001),reaching the peak at the first day and gradually down to the normal level at the sixth day.MFGE8 was gradually increased in CCl4-induced chronic injury(p < 0.001).Real-time PCR analysis showed that the expression of MFGE8 was increased in CCl4-induced acute injury by 4.4-folds at the third day(p < 0.001),and increased in CCl4-induced chronic injury by 4.2-folds and 5.0-folds at the second week and the sixth week,respectively(p < 0.001).Increased expression of MFGE8 was also confirmed by western blot.To further examine the secretion of MFGE8 from hepatocytes,primary mouse hepatocytes were isolated.Results indicated that the secretion of MFGE8 was increased along with the culture time and the CCl4 exposure further increased the expression of MFGE8.Increased expression of MFGE8 was also confirmed by western blot and real-time PCR(p < 0.05).When primary mouse hepatocytes were induced by CCl4,Bcl-2 expression was decreased and Bax expression was increased;while rmMFGE8 protein was added,the increased expression of Bcl-2 and decreased expression of Bax indicated the anti-apoptosis role of MFGE8.When LX-2 cells was treated with rhMFGE8,the activation of LX-2 was suppressed in a dose-dependent manner.In the situation of Huh-7 cells transfected with siRNA,MFGE8 expression was suppressed,along with the decreased expression of Bcl-2 and increased expression of Bax,which indicated the anti-apoptosis role of MFGE8 in Huh-7 cells.Part 3 Potential clinical significance of MFGE8 in hepatitis-liver cirrhosishepatocellular carcinoma progression.The expression of MFGE8 in normal human tissues was examined by immunohistochemical staining,indicating that MFGE8 was expressed widely in epithelium-originated cells.To further clarify the clinical significance of MFGE8 in hepatitis-liver cirrhosis-hepatocellular carcinoma progression,serum and tissues from hepatitis,liver cirrhosis and hepatocellular carcinoma patients as well as healthy donors were examined by ELISA and immunohistochemical staining.Immunohistochemical staining validated that MFGE8 was expressed in both cytoplasm and cell membrane of hepatocytes.The expression of MFGE8 was significantly decreased in hepatocellular carcinoma tissues(p < 0.01).ELISA results indicated that serum MFGE8 expression was increased in hepatitis patients(p < 0.01),while decreased in liver cirrhosis and hepatocellular carcinoma patients.Potential diagnostic and therapeutic values of MFGE8 need to be further investigated.