| BackgroundPrimary hepatic carcinoma is the second most common cancer in the world and is one of the most common invasive tumors.According to the International Cancer Center IARC,there are about 850,000 new cases of hepatic carcinoma every year.Hepatocellular carcinoma（HCC）is the absolute main type of primary hepatic carcinoma.Although advances in HCC treatment technology have significantly prolonged the survival of patients with hepatic carcinoma,the mortality rate of HCC patients remains high.Metastasis is a common problem in the treatment of HCC,and it is also the main cause of death in hepatic carcinoma patients.At present,the common clinical use of surgery,radiotherapy and chemotherapy are difficult to achieve the desired therapeutic effect on metastatic HCC.Hepatocarcinoma metastasis is a complex process involving multiple steps and multiple factors,and is regulated by a variety of signaling pathways and related molecules such as cell proliferation,invasion,movement and adhesion.Although HCC metastasis has been a research hotspot at home and abroad,the specific mechanism of its occurrence has not yet been fully elucidated.Iron is an indispensable element for all living things and is involved in the process of oxygen transport and utilization.Many studies at home and abroad,including the previous research of our research group,found that iron content was significantly reduced in cancer tissues of hepatic carcinoma patients and chemical agents induced by primary hepatic carcinoma in mice.Even in HCC caused by iron overload,the iron content of cancer tissue is lower than that of adjacent tissues.Significantly,our group found that low-iron promoted the metastasis of Hepatocarcinoma cells through in vitro and in vivo experiments.Therefore,we believe that exploring the mechanism of low iron production in liver cancer tissues is of great significance for the prevention and treatment of HCC metastasis and the monitoring of the prognosis of HCC patients.ObjectiveBy observing the changes of iron content and iron metabolism protein expression in liver cancer patients,experimental animal cancer tissues and normal（adjacent to cancer）liver tissues,the key iron metabolism protein that may lead to low iron in liver cancer tissues was analyzed,and the effect of this protein expression change on iron homeostasis in liver tissues and even the whole body was confirmed by in vivo and in vitro experiments.To provide clues for the comprehensive understanding of the causes of low iron in liver cancer tissues and the development of corresponding prevention and treatment measures.MethodsⅠ.Cell Experiment1.Cell cultureHuman hepatoma cells Huh7,HepG2 and mouse hepatoma cells Hepa1-6 were cultured in high glucose DMEM medium（Hyclone）containing 10%fetal bovine serum（Gibco）and 1%double antibody（Gibco）;hepatocytes L02 were cultured in 10%fetal calf serum and 1%double-antibody 1640 medium（Sigma）;human primary hepatocytes cultured in 5%fetal bovine serum（Sciencell）,1%double antibody（Sciencell）and 1%hepatocyte growth additive（Sciencell）in hepatocyte culture medium.The cells were placed in an incubator at 37°C in a 5%carbon dioxide solution.2.Cell processingThe iron chelator DFO was used at a concentration of 0,50,100,200μM and the intervention time was 24 hours.The heme was used at a concentration of 30μM and the intervention time was 24 hours.Holo-Tf was used at a concentration of 30μM and the intervention time was 24 hours.3.Detection of intracellular iron contentThe intracellular iron content was measured using Biovision’s iron colorimetric assay kit（K390-100）.4.Cell transfectionThe interfering RNA and overexpression plasmid were transfected with Lipo3000.The concentration of interfering RNA was 100μM,and the concentration of overexpression plasmid was 1 ug/ul.The specific transfection dosage and procedure were as described in Lipo3000,and the transfection time was 48 hours.Ⅱ.Animal Experiments1.Animal breedingC57 mice,Wistar rats,and feeding feeds were purchased from the Animal Center of the Second Military Medical University,and the experimental procedures followed the human care standards for experimental animals.The feeding temperature is maintained at around 25°C and the humidity is around 50%.The day and night light alternates.Keep the feeding environment clean and hygienic.2.Rat hepatocellular carcinoma modelSeven-week-old male Wistar rats were divided into two groups.The control group consisted of 6 rats:PBS was intraperitoneally injected with 0.5 ml once a week.In the experimental group,10 rats were intraperitoneally injected with DEN 0.5 ml at a dose of50 mg/kg once a week.Continuous injection for 16 weeks,additional two weeks of blank elution period and sacrifice.3.Mouse hepatocellular carcinoma modelTwo-week-old male C57 mice were randomly divided into control group and DEN group.The control group received a single dose of 0.1 ml intraperitoneal injection of PBS.The mice in the experimental group were intraperitoneally injected with 0.1 ml of DEN at a dose of 25 mg/kg body weight.From the day of intraperitoneal injection,the mice were sacrificed in1m,3m,5m,7m,and 9m to ensure at least 6 mice per group at each time point.4.Liver HCP1 interference,overexpression mouse modelSixteen six-week-old C57 male rats were randomly divided into four groups:a blank control group,a negative control group,a liver-specific interference HCP1 group,and a liver-specific overexpressed HCP1 group,four in each group.The virus was injected through the tail vein at a dose of:negative control virus:5 x 1011vgs;HCP1interfering virus:2 x 1012vgs;HCP1 overexpressing virus:1 x 1012vgs.It was sacrificed6 weeks from the date of injection of the virus.5.Detection of serum related indicatorsThe blood was condensed for half an hour at room temperature,centrifuged at 3000g for 20 min at 4°C,and the serum was carefully aspirated.Serum iron,transferrin saturation,total iron binding capacity were measured by automatic biochemical analyzer;serum alpha-fetoprotein was detected by ELISA kit,and heme was detected by QuantiChrom kit.6.HE and GFP staining of rat liver tissueⅢ.Protein extraction and expression detection of tumor tissues and adjacent tumor tissues of patients with liver cancerThe whole protein extraction kit was used to extract the whole protein of 12 cases of HCC patients from tumor tissues and adjacent tissues,and the protein expression level of iron metabolism related molecules was detected by Western Blot.Ⅳ.Statistical methodsThe experimental data in the graph is presented as Mean±SEM.The data of each group were consistent with the variance,the t test was used for comparison between the two groups,and the variance analysis was used for multiple groups.If the data of each group did not meet the homogeneity of variance,the Mann-Whitney test was used for comparison between the two groups,and the Kruskal-Wallis test was used for comparison between groups.When the P value was<0.05,the difference was considered to be statistically significant.ResultsⅠ.Iron homeostasis and expression of regulatory molecules in HCC tissues and hepatoma carcinoma cells1.Iron content and iron metabolism protein expression in human liver cancer and paracancer tissuesMany domestic and foreign studies,including our research group,found that the iron content of liver tumor tissues was lower than that of adjacent tissues.In order to explore the causes of decreased iron content in liver tumor tissues,we examined the expression levels of a series of iron metabolism-related molecules in tumor and adjacent tissues.Compared with adjacent tissues,the expression of TFR1 and FPN were increased and Hepcidin was decreased in Hepatocarcinoma tissues;the expression of Steap3,DMT1 and HCP1 were decreased.2.Iron content and iron metabolism protein expression in liver cancer and normal liver tissue of ratsWe established a rat HCC model by intraperitoneal injection of DEN,and found the formation of HCC by visual observation of tumor nodules and HE staining to indentify the morphology of liver cells.Compared with normal liver tissues,the iron content of tumor tissues decreased and the expression of TFR1 were increased;Hepcidin,FPN,Steap3,DMT1 and HCP1 were decreased.3.Changes of hepatic iron content and expression of iron metabolism protein during HCC formation in miceWe induced HCC in mice by intraperitoneal injection of DEN.Five consecutive time points were set during the formation process for dynamic observation.The results showed that no liver tumors were formed in the DEN group at the time of 1,3,and 5months.At time of 7 months,some mice in the DEN group formed liver tumors,and all mice in the DEN group formed liver tumors at the time of 9 months.The liver iron content and the expression of liver iron metabolism molecules in mice injected with DEN for 1,3,and 5 months were not significantly different from those in the control group at the same time.Compared with adjacent tissues and normal liver tissues,the iron content of liver tumor tissues was significantly decreased in the intraperitoneal injection of DEN mice for 7,9months and expression of TFR1 were increased;Hepcidin,FPN,Steap3,DMT1and HCP1 were decreased.4.Expression of iron metabolism protein in iron deficiency experiment of liver cancer cellsIn order to explore the causal relationship between the expression levels of iron metabolism related molecules and decreased iron content in hepatocarcinoma tissues,we added DFO to HepG2 and Huh7 cells,and observed that expression of TFR1,FPN,Steap3,DMT1 and HCP1 were elevated in the absence of iron in cells.Hepcidin was decreased.Ⅱ.HCP1 affects liver tissue cells and even systemic iron homeostasis in vitro and in vivo experiments1.Holo-Tf culture was conducted after interfering with Steap3,DMT1 and HCP1and the intracellular iron content remained unchangedCompared with the control group,hepatocytes interfered with Steap3,DMT1 or HCP1 and added transferrin-binding iron for 24 hours to detect intracellular iron content.The results showed that there was no significant difference of iron content between the two groups.Since Steap3 and DMT1 are located upstream and downstream in the pathway of transporting Holo-Tf,we also added Holo-Tf culture after interfering with Steap3 and DMT1 at the same time.There was no significant difference of iron content between the two groups.2.Heme culture was conducted after interference and overexpression of HCP1 and the intracellular iron content was changedHeme is an important form of iron in the body,and HCP1,as a newly developed heme transporter,is expressed in the liver,but whether it can transport heme is still unknown.Our team found that hepatocyte L02 can take heme through HCP1.In this experiment,we further expanded the cell lines and added heme to human primary hepatocytes and hepatoma cells Huh7,HepG2,and Hepa1-6 for 24 hours,the intracellular iron content was increased.On this basis,we used SiRNA to interfere with HCP1 and added heme for 24 hours,the results showed that the iron content of the interference group was lower than that of the control group.After over-expressing HCP1 by plasmid and then heme was added for 24 hours,the intracellular iron content was increased compared with the control group.3.The expression of hepatic HCP1 affected liver and systemic iron homeostasisBy detecting the expression of GFP and HCP1,we confirmed that HCP1 was specifically knocked down or overexpressed in mouse liver.Compared with the blank control and the negative control,the liver iron content and the Hepcidin protein level of the SiHCP1 group decreased.The expression of membrane transferrin FPN is elevated,consistent with the performance of low iron in the liver.Serum results showed that iron,transferrin saturation,and heme increased in HCP1-interfering mice.However,the liver iron content,serum iron level and the expression level of iron metabolism-related molecules in liver tissues of OverHCP1 group were not significantly different from those of the control group.ConclusionThis study by comparing iron contents and the expression of iron metabolism proteins in liver cancer tissues and paracancerous tissues of human,rat and mice,confirming that cancer tissues has lower iron content.We observed consistent characteristics of iron metabolism protein expression in three different species of liver cancer tissues:compared with normal liver tissue and paracancerous tissues,cancer tissue has higher TFR1 and FPN expression;The expression of Hepcidin,Steap3,DMT1and HCP1 decreased.In addition,we observed a gradual decrease in hepatic iron content and altered expression of iron metabolic protein during the formation of HCC in mice.Combined with the iron deficiency experiment of liver cancer cells,the decrease of Steap3,DMT1 and HCP1 in the cancer tissues could not be explained by the low iron level of liver cancer.Since they are all involved in iron uptake in cells,we further confirmed at the cellular level that interfering with the expression of these three molecules would not change iron content of cells by affecting the transport of holo-tf.HCP1,as a heme transporter,Whether can transport hepatocyte heme is still unknown.Therefore,we confirmed the regulatory effect of HCP1 on the iron content of hepatocyte and the systemic iron homeostasis by combining in vitro and in vivo experiments after it was clear that hepatocyte（cancer）cells can absorb heme.Furthermore,it was speculated that the decreased expression of HCP1 in liver cancer tissues might be an important reason for the low iron level in cancer tisuues.Our study lacks direct evidence to prove that the reduced expression of HCP1 in cancer tissues is the cause of low iron in liver cancer.Due to the time,this part of the experiment will be carried out in the future. |
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