Ferroportin Inhibits Hepatocellular Carcinoma Growth Mechanism Research

Objective:The Iron is essential material to maintain normal cellular function. Many malignant tumors showed increased demand for iron, probably the iron can be used as a certain proteins involved in maintaining of cell growth and proliferation.Ferroportin is called the member1pritein of solute transporter family40, which mainly expressed in the mesh of the hematopoietic system, duodenum, spleen, liver, kidneys and heart, and the embryonic ectoderm also expressed. These systems is closely related to iron metabolism. The subcellular localization indicated that Ferroportin mainly expressed on the cell membrane and cytoplasm, including liver cells, glomerular epithelial cells, macrophages, duodenum epithelial cells. The main function of Ferroportin is transport iron from intracellular to extracellular,which can maintain the balance of iron metabolism and absorption. But Ferroportin only transported intracellular iron out of the cells, the opposite transportion is responsible for other proteins.Ferroportin key material of iron metabolism, that can control the level of intracellular iron.The expression levels of Ferroportin regulatied by at least three mechanisms:the transcriptional regulation, Translation regulation and Protein level regulation. Protein level of regulation is mainly manifested in combining hepcidin Specifically,and then ferroportin degradated. It is comfirmed that Hepcidin is the sole ligand of Ferroportin. Ferroportin phosphorylated after the combination of hepcidin and Ferroportin,which can mediate internalizated of Ferroportin, and degradated by the proteasome.Hepatocellular carcinoma (HCC) is a highly malignant tumor, the malignant tumor is one of the serious threat to human life and health. The Morbidity of HCC in our country ranked forth in magient tumor,that is every one hundred thousand population was about23.5, and the death rate of HCC ranked second. corresponding of the developmeng the infection by HBV and HCV, The incidence of HCC in western countries are growing. So far,many research indiated that many genomic aberrations of expression exist in HCC.Ferroportin expressed highly in the liver cells, and the liver is the key organ of iron metabolism. In this study, we detected the expression levels of Ferroportin protein of clinical cases specimens in patients with HCC, further experimental study of cell biology Ferroportin protein and liver cancer cell malignant behavior, also molecular biology experiment to study the specific molecular mechanism; and preliminary clarify the mechanism and significance of Ferroportin protein in the development of HCC.Method:Ferroportin was detected by the immunohistochemical staining in60cases of HCC,corresponging adjacent liver tissue and10cases of normal tissure,at the same time the relationship of the Ferroportion expression and Clinical and pathological features also analyed. Further more through the constrcution of over expression cell lines,the expression of Ferroportin protein improved in Liver cancer cell line MHCC-97H. Using growth curve, MTT experiment, scratch test, Matrigel invasion of experimental to study of Ferroportin protein on high metastatib HCC cell biology behavior in rats, and the fluorescence measurement method for the detection of the labile iron pool in Ferroportin cells (LIP) effect.Finally, quantitative PCR and Western confirmed the reason of low Ferroportin expression in hepatoma cells.Result:The immunohistochemistry results showed that:(1)In the cyt oplasm and cell membrane of normal liver tissue adjacent tissues,Ferropo rtin have shown a strong positive expression. Only a small part of HCC ti ssues was weakly positive expression. The statistical analysis of Ferroport in protein-positive rate was28.3%(17/60) and the score was0.96±0.31i n the HCC tissues; The Ferroportin protein expression positive rate was91.17%(55/60), and the score was5.97±2.12in the adjacent tissues; the Ferroportin protein expression positive rate was100%(10/10), and the sc ore was6.38±2.59in the normal liver tissue.The statistical analysis of the relationship between expression levels and their clinicopathological features of60cases of HCC patients Ferrop ortin Show:Ferroportin protein positive expression rate was16.67%, and t he positive score was0.69±0.33in the hepatocellular carcinoma patients associated with intrahepatic metastasis; the positive expression rate was31.25%, and the positive score was1.03±0.43, in the hepatocellular carc inoma patients negetive associated with intrahepatic metastasis,the differ ence of the two groups have statistical significance P<0.05. Ferroportin p rotein positive expression rate was9.10%, and the score was0.72±0.28wich have portal vein invasion; the positive expression rate was66.67%, and the score was1.03±0.47which not associated with portal vein invas ion,the difference between the two groupa have statistically significant, P <0.05. the relationship of the Ferroportin protein expression and Edmond son-Steiner grade of hepatocellular carcinoma cells:in the Ⅲ-Ⅳ group, t he Ferroportin protein positive expression rate was20.00%, and the positi ve score was0.73±0.32;in the Ⅰ-Ⅱ group, the positive expression rate w as35.29%, and the positive score was1.14±0.49, the difference between the two groep have statistical significance P<0.05. TNM staging of hepat ocellular carcinoma and the Ferroportin protein expression have existence a similar relationship. In the III-IV group, the Ferroportin protein expres sion rate was19.23%, and the positive score was0.73±0.33; In the1Ⅰ to Ⅰ I group, the positive expression rate was35.79%, and the positive score w as1.15±0.45, The difference between the two group have statistical sign ificance P<0.05. the expression of Ferroportin protein and AFP levels ha ve no obvious relationship P>0.05Expression of Ferroportin in hepatoma cell lines, normal liver cell line:Western-blot experiment showed:Ferroportin in hepatocellular carcinoma cell lines expressing significantly decreased, and normal liver cell line is increased expression, there were significant differences p<0.05, and liver cancer cell line MHCC-97H expressed Ferroportin in the lowest level. Construction of eukaryotic expression vector of Ferroportin, and identification and verification of the Western-blot was transfected into MHCC-97H cell line.Growth curve experiments show that:transfected by Ferroportin, first to4days of cell growth were:16750±5212,23445±6198,48377±4768,91642±7698, and the difference to control group and untreated group was significant, p<0.05. The control group of first to4days of cell growth were:21563.9±5860,47344.4±6379,89561.1±4870,155862±7980; untreated group of first to4days of cell growth were:22342±5490,45244±6523,90124±5062,149782±7120, the differences between no statistical significance, P>0.05.The experimental results of MTT show:Ferroportin transfected group0-96hours of light absorption rates were:0.175±0.0447,0.267±0.0451,0.437±0.0567,0.765±0.0788,1.134±0.0853; while the control group24-96hours of light absorption rates were:0.198±0.0479,0.356±0.0675,0.872±0.0345,1.421±0.0933,1.748±0.1012; the untreated group,24-96hours of light absorption rates were:0.202±0.0564,0.362±0.0764,0.843±0.0671,1.453±0.1392,1.752±0.1132. the differences were statistically significant between Experimental group, untreated group and the control group, P<0.05, and untreated group compared with the control group there was no significant difference, P>0.05Inhilbition rate curve dispay:untreated group and control group each time training the inhibition rate difference were not statistically significant (P>0.05); three groups of cells cultured for0hours in the inhibition rate of no statistical significance (P>0.05), the experimental group and the other two groups of inhibiting cell strain rate between24hours after each time point suppression comparison of the differences were statistically significant (P<0.05),96h was72hours compared with inhibition rate increased, but there was no significant difference between the two (P>0.05). And incubated for72hours when the inhibition rate was31.12±7.87, the inhibition rate of>0.3, drug sensitive test positive.The scratch test experiment shows:Ferroportin transfected group36hours of cell movement distance is268±28.94microns, while untreated group and the control group is483±24.08,479±23.18micron micrometer. There were statistical significance between the experimental group and untreated group, P<0.05.Matrigel invasion assay revealed:untreated group, control group in MHCC-97H cells through the basement membrane of the cell number for each visual field mean172±33.43,162±30.43; and Ferroportin after transfection, MHCC-97H cells through the basement membrane of the cell number decreased significantly, each visual field mean28±10.67, experimental group and untreated group, in comparison to the control group, the difference has statistical difference, P<0.01.Fluorescence measurement method for the detection of intracellular labile iron pool (LIP) the results showed that:in the untreated group, the control group MHCC-97H LIP were0.3857±0.086,0.3746±0.091rfu; MHCC-97H cells in experimental group LIP is0.1238±0.076rfu. The experimental group and the control group, control group, two proups showed significant difference, P<0.01.Western-blot detection of Hepcidin on Ferroportin expression and intracellular labile iron pool in LIP effects show:(1) using the0nM,300nM,700nM concentration of Hepcidin transfected with Ferroportin MHCC-97H cells, the expression levels of Ferroportin were1.102±0.457,0.479±0.213,0.164±0.087,300nM,700nM and Ogroup nM expression level of Ferroportin had significant difference, p<0.05.3O0nM and700nM groups had significant difference, p<0.05.(2) the determination of0nM,300nM,700nM concentration of Hepcidin in experimental group MHCC-97H cells in LIP,0nM,300nM,700nM Hepcidin after treatment with LIP were0.1312±0.069,0.2576±0.081,0.3913±0.072300nM,700nM and OnM group LIP had a significant difference, p<0.05300nM and700nM groups had significant difference, p<0.05.RT-PCR detection of BMP/Smad signaling pathway on the expression of Ferroportin (1):the effects of different kinds of factor BMP on the expression of Hepcidin results show is not using the BMP treatment, the expression quantity of Hepcidin is0.978±0.145; BMP2, BMP4, BMP6after treatment with Hepcidin expression ratios were:13.2600±1.2450,12.9340±1.3670,14.2300±2.1560; and the use of BMP inhibitors in group Hepcidin expression ratio was0.0560±0.2360. BMP2, BMP4, BMP6after treatment with Hepcidin expression and the unused BMP treatment BMP inhibitor Hepcidin expression, there were significant differences between, P<0.05,Fluorescence quantitative PCR detection of BMP2different treating time Hepcidin expression results show:BMP2treatment after0.51,2,4,6,12hours of Hepcidin expression ratio were1.423±0.356,2.534±0.215,5.871±0.534,8.314±1.098,14.23±1.230,18.240±1.780, each time point comparison between the differences were statistically significant, P<0.05.Western-blot detect the Ferroportin expression in MHCC-97H cell lines treated by BMP2、BMP4、BMP6:The use of BMP2, BMP4, BMP6MHCC-97H cells treated with the signaling pathways downstream of phosphorylation of Smad (1/3/5) phosphorylation increased significantly, While the use of BMP inhibitor Smad (1/3/5) phosphorylation were significantly inhibited, BMP2, BMP4, BMP6after treatment with Hepcidin expression and the unused BMP treatment BMP inhibitor Ferroportin expression, there were significant differences between. But BMP family member2,4.6deal with expression of Hepcidin showed no significant differences.Conclusion:1.The protein Ferroportin has low expression and defection in HCC,related with Edmondson-Steiner grading, TNM stage, Liver metastasis and Portal vein invasion.2.Protein Ferroportin has low expression in Hepatocellular carcinoma cell lines,the result is opposite in normal cell lines.3.The Construction of eukaryotic expression vector of Ferroportin is succeed.4.In MHCC-97H cell lines,the high expression of Ferroportin can Obvious inhibite the Growth and invasion metastasis of MHCC-97H cells.5. The high expression of Ferroportin can make the hepatoma cell intracellular iron concentration decreased significantly.6. Hepcidin can promote degradation of Ferroportin cell, making Ferroportin expression decreased, which rise the iron concentrations in hepatocellular carcinoma cells. 7. The members of the BMP family of2,4,6can promote the increasion of Hepcidin expression, which can ruduce the expression of Ferroportin.