Iron Deficiency Promoted Metastasis Of Hepatoma Cells Involving Upregulation Of SPNS2

BackgroundPrimary hepatocellular carcinoma?HCC?has a high incidence and poor prognosis in China,with the second mortality rate in all types of cancers.the risk factors of HCC in China,even in East Asia,are hepatitis B virus infection and exposure to aflatoxin B1 in environment or food.Although several studies have reported that iron-overload in liver is an independent risk factor for HCC,the tumor tissues of HCC appears to have decreased iron content in comparison to adjacent tumor tissues or normal tissues.Even in HCC induced by iron-overload,there is also a decrease of iron content in HCC tumor tissues and exhibit iron-free tumor nodules.The reasons for the decrease of iron content in HCC are still unclear.As the essential elements needed for cell growth and proliferation,the lack of iron inhibits cell proliferation.therefore,Iron Chelator is applied to cancer treatment,including HCC.But the malignancy of cancer is largely dependent on metastasis,because more than 90%cancer patients eventually died from metastasis.At present,it is unclear whether the iron deficiency in tumor tissues plays a role in the metastasis of HCC.Hence,based on the decrease of iron content in HCC tumor tissues,we prove that iron deficiency promotes migration and invasion of hepatocellular carcinoma cells in vitro,as well as metastasis in vivo,but the mechanisms need to be further explored.SPNS2 is a transporter of sphingosine-1-phosphate?S1P?,which is an immune-regulated molecular.It is reported that SPNS2 knockout does not change the content of S1P in circulation but has an effect on function and survival of T cell.A recent study published in Nature shows that SPNS2 knockout has the most significant role in inhibiting melanoma lung metastasis from the 810 kinds of gene knockout mice.SPNS2^-/-mouse shows a decrease of lymphocytes in circulation and an increase of effects T cells and NK cells in lung,which leads to inhibition of tumor metastasis.Meaningfully,we found that SPNS2 expression was increased by iron deficiency in HCC cell lines,meanwhile the level of SPNS2 expression was also elevated in hepatocellular carcinoma.Thus,we wonder that does iron deficiency play a role in promoting the metastasis of hepatocellular carcinoma via upregulation of SPNS2?Will the SPNS2 knockout effectively block the promotion of iron deficiency on the metastasis of hepatocellular carcinoma?And what is the mechanism of iron deficiency to increase the expression of SPNS2?These questions have yet to be studied.ObjectiveThis study compared the iron content in tumor tissues with adjacent tumor tissues of HCC and the iron content in tumor tissues with metastasis with tumor tissues without metastasis to analyses the relationship between iron content with HCC metastasis.Then we clarified the role of iron deficiency on HCC metastasis to help us understand the reasons of poor prognosis and high rate of metastasis and recurrence of HCC and provide theoretical guidance for clinical use of iron chelator in the treatment of hepatocellular carcinoma.MethodsI.Detection of HCC tissue samples1.Detecting iron content by MRIWe used GE Signa HDX 1.5T MR scanner to scan the HCC patients according to the set parameters before surgical operation.Then used workstation software to build R2*map.After manually marked the area of interest?ROI?in R2*map,we exported the R2*value analyzed by software.2.Flame Atomic absorption spectrometryAfter weighted the dried cutting tissues,we placed them in digestive tube with addition of 1ml mixed acids from nitric acid and perchlorate?1:4?.Used high-temperature digestion furnace to digest tissues till solution clarification,and then set to 10ml.The standard curve sample was configured from standard product Iron Standard stock liquid?100?g/ml?.The iron content in the solution was calculated according to the standard curve,and then the iron content in the tissues??g/g?was obtained.?.Cell Experiment1.Cell culture and interventionHuh7 cells and HepG2 cells were cultured in high glucose DMEM containing 10%fetal bovine serum and 1%double antibiotics,SMMC-7721 cells and H22 cells were cultured in RPMI-1640 containing 10%fetal bovine serum and 1%double antibiotics.Iron chelator DFO was used at a concentration of 100?M,and Dp44mT at 10?M for 24 hours.HIF1?inhibitor PX478 was used at a concentration of 20?M,SP1 inhibitor Mithramycin A was used at 100nM.2.Primary hepatocytes ExtractionHuman primary hepatocytes were purchased from Sciencell with an identification report.Mouse primary hepatocytes extraction:After taking out the liver,tattered it with surgical scissors,then added trypsin-EDTA with blowing and suction.Used 400 mesh to filter the cell suspension and transferred to cell culture bottle.At last,washed away the un-adherent cells next day.3.Cell proliferation detectionWe used the ThermoFisher Click-iT?Plus EdU Alexa Fluor?488 Flow Cytometry Assay Kit to detect cell proliferation.4.Migration and Invasion experimentWe used transwell chambers to detect the migration ability of HCC cells.The changes of migration ability of HCC cells under iron deficiency were quantitatively analyzed by using the CytoSelect?96-Well Cell Migration Assay Kit.We used matrigel precoated transwell chambers to qualitatively detect invasive capability of HCC cells.The invasion ability of HCC cells was quantitatively analyzed by using CytoSelect?96-Well Cell Invasion Assay kit.5.TransfectionWe used Lipofectamine^?3000 for siRNA transfection and followed the instructions.The medium was replaced at next day and cells were proceeded to next operation after 48hours.6.Label of luciferase in cellsWe used luciferase labeling kit to label cells with luciferase.The kit contains lentivirus mediated luciferase marker and assisted infection solution Polybrene.7.Knockdown of TFRC in cellsWe used the lentivirus carrying TFRC shRNA to knockdown the transferrin receptor of Huh7 cells and H22 cells.8.Fluorescence quenching assay for iron content in cellsWe used Phen Green^TM FL reagent?Thermo Fisher?to observe the contents of iron in cells.9.Transcription factor activity chipExtracted nuclear protein from Huh7 Cells after intervention with DFO for 24h.incubated with 345 kinds of probes in the Transignal^TM Protein/DNA Array Kit which specificly bind with the transcription factor.Then washed away the unbound probes and isolated the probes from protein/DNA complex.Hybridized probes with the transignal chip and detected the signal value of each probe to reflect the binding activity of the transcription factors.10.Chromatin immune coprecipitationWe used CST SimpleChIP kit to detect binding activity of three transcription factors HIF1?,SP1 and YY1 with SPNS2 promotor.?.Animal experiments1.Animal feeding and groupingAnimal experiments are approved by the Ethics Committee of the second Military Medical university and follow the norms of humane care in animal experiments.C57 mice,nude mice,and TFRC knockout mice,SPNS2 knockout mice were reared in the SPF room.Groups of C57 in vivo imaging experiments:12 male C57 mice aged 5-6 week were randomly divided into two groups:liver in situ tumor-bearing group and caudal vein tumor-bearing group.Each group was divided into two subgroups:normal diet group and iron deficiency diet group?n=3?.After feeding the iron deficiency diet for one week,we planted firefly-luciferase marked H22 cells in situ of liver or via caudal vein.Every 10 days,we recorded in vivo images of tumor cells.H22-fluc-TFRC^knockdown in vivo experiment:12 male C57 mice aged 5-6 week were randomly divided into two groups:liver in situ tumor-bearing group and caudal vein tumor-bearing group.Each group was divided into two subgroups:H22-fluc group and H22-fluc-TFRC^knockdown group?n=3?,all groups were fed with normal diet.Huh7 cells were planted in nude mice and grouped same as C57 mice.Group of NCG mice:two models of iron deficiency in mice and intracellular iron deficiency were included.Each model contained liver in situ implantation of Huh7 cells and H22 cells?n=3?.TFRC knockout mice were divided into two groups:heterozygote group?4 male and 2 females,n=6?and matching wildtype mice group.Both groups were fed normal diet and liver in situ implantation of H22-fluc cells at 6 weeks age.In vivo images were recorded every 10 days.Group of SPNS2 knockout mice:2 groups of liver in situ tumor-bearing group and caudal vein tumor-bearing group with 2 subgroups in each group:knockout group?3 male?and same nest wildtype group?3 male?.Both groups were fed iron deficiency diets and planted H22-fluc cells in situ or caudal veins at 6 weeks age.2.Liver in situ tumor-bearing and caudal vein tumor-bearingThe cells were mixed with Matrigel to form a cell suspension?2 x 10⁷/ml?.An aseptic micro incision was cut along the lower edge of the left rib at the lower edge of the left rib after anesthesia in the mice to show out the liver.The 0.1ml cell suspension was extracted and slowly injected with 30 degrees.After the injection,pressed with gelatin sponge for 30s,the abdominal muscles and the skin were sutured layer by layer.The caudal vein was injected with a slow small volume injection of the tail vein.3.In vivo imagingMice were photographed every 10 days,until 5 times if no mice died.After the mice were anesthetized,the luciferase substrate was injected into the abdominal cavity.After the substrate was spread,the photo was taken and analyzed with the instrument analysis software.4.ImmunofluorescenceImmobilized mouse tissue.embedded in paraffin.cut sections and dewaxed.After antigen repair,incubated antibody,then used DAPI staining the nucleus.Used anti-fluorescence quenching agent seal the slices,pictured by a fluorescent inverted microscope.5.Serum iron indicatorsThe serum iron content,transferrin binding capacity?TIBC?and transferrin saturation?TS%?were obtained by Hitachi 7600 Automatic Biochemical Analyzer.Serum ferritin content was detected by Elisa kit.6.Construction and identification of TFRC knockout miceTFRC knockout mice were purchased from Nanjing Institute of Biomedical Sciences,Nanjing University.The exon3 in TFRC gene was deleted 202bp leading to a shift code mutation.7.Construction and identification of SPNS2 knockout miceWe commissioned the Cyagen Biosciences Inc to construct SPNS2 knockout mice.The exon3-5 in SPNS2 gene were knockout.8.Detection of lymphocyte subsets proportion by flow cytometryFirst,we extracted total white cells from tissues by using white cell extraction kit.Then we incubated the white cells with antibody binding with lymphocyte surface antigen.Through detecting the fluorescence of marked white cells by flow cytometry,we obtained the proportions of wanted lymphocyte subsets in white cells.IV.StatisticsWe represented the data as Mean±SEM.The t-test was used for two-group comparisons,and one-way ANOVA was used for multigroup comparison,if the data obeyed the normal distribution.If the data did not obey the normal distribution,the Mann-Whitney test was used for two-group comparisons,and the Kruskal-Wallis test was used for multigroup comparisons.Differences were considered significant at the 95%confidence level?p<0.05?.ResultsI.The iron content of tumor tissues was decreased and lower in metastatic HCC tissues than non-metastatic HCC tissues.1.Decreased iron content in liver cancer tissueThe results showed that the R2*value of tumor tissues was significantly lower than the R2*value of adjacent tumor tissues and distant normal liver tissue.Since the R2*value is positively correlated with iron content in the multiple-echo R2*MRI scan,we thought preliminarily that the iron content of tumor tissues was lower than that of adjacent tumor tissues and distant normal liver tissue.We collected the resected tumor tissues and isolated adjacent tumor tissues.The iron content in tumor tissues was significantly lower than that of the adjacent tumor tissues by FAAS,which confirmed the MRI results.we found that changes of TFRC,HAMP and Ferritin expression were coincidence with appearance of iron deficiency.Therefore,it was reasonable to believe that the iron content of tumor tissues was lower than adjacent tumor tissues and distant normal liver tissue.2.The iron content of tumor tissue in metastatic HCC tissues was significantly lower than that in non-metastatic HCC tissues.A total of 15 patients with metastatic HCC and 15 patients without metastatic HCC were collected 5 years after surgery.By detecting the iron content and the expression of iron metabolism molecules,we found the iron content of metastatic HCC tissues was significantly lower than that without metastasis.II.Iron deficiency promoted the metastasis of hepatocellular carcinoma cells in various models both in vitro and in vivo.1.Iron deficiency inhibited proliferation of hepatoma cells but promoted migration and invasion.We used DFO at 100?M and DP44MT at 10?m to chelate cell iron.Then we used flow cytometry to detect the effects of iron deficiency on proliferation of three HCC cells,Huh7,HepG2 and SMMC-7721.The results showed that the proliferation of three kinds of HCC cells was strongly inhibited by iron chelators.We found that three kinds of HCC cells showed significant increase of migration and invasion abilities under the action of two kinds of iron chelators by using qualitative and quantitative detection methods.2.Iron deficiency in nude mice promoted metastasis of Huh7 cells.We first fed nude mice with iron-deficient diet to observe effects of iron deficiency on metastasis of Huh7 cells in vivo.We confirmed that the iron deficiency model of nude mice was successfully constructed by detection of iron content in liver and serum,transferrin saturation in serum,and immunofluorescence of ferritin in liver and lung.Huh7 cell was labeled with firefly-luciferase?Huh7-fluc?and cultured to a sufficient number for implantation in situ in nude mice liver.It was found that the iron deficiency group showed obvious enhancement of metastasis compared with normal group,which hinted that iron deficiency facilitated metastasis of Huh7 cells in vivo.it was found that the tail intravenous injected Huh7-fluc cells formed tumors mainly in the lung,and the areas of tumors in iron deficiency group was significantly larger than that in the normal group.3.TFRC knockdown in Huh7 cells caused intracellular iron deficiency and promoted its metastasis in nude mice.In order to fit the phenomenon that iron deficiency in HCC tissues not in the whole body,we planned to cause iron deficiency in cells by knockout the transferrin receptor gene in Huh7 cells,which restricted its ability to transport iron.Detection of TFRC mRNA expression and TfR1 protein expression displayed successful knockdown of TFRC in Huh7-fluc cells and the content of iron in knockdown cell was lower than that in wildtype cells.Huh7-fluc-TFRC^knockdown and Huh7-fluc were implanted in the liver of the nude mice with the same amounts of cells,and all nude mice were fed with normal diet.The results showed that the metastasis of Huh7-fluc-TFRC^knockdown cells was significantly enhanced and the pulmonary metastasis was significant compared to Huh7-fluc cells in nude mice.Similarly,the metastasis of Huh7-fluc-TFRC^knockdown cells in the nude mouse was increased than Huh7-fluc cells by tail vein injection,which meant intracellular iron deficiency could promote the metastasis of hepatoma cells in vivo.4.Iron deficiency in C57 mice promoted metastasis of H22 cells.we implanted H22 cells in C57 mice to observe effects of iron deficiency on metastasis of H22 cells in vivo,in order to eliminate effects of immune system defects on the metastasis of hepatocellular carcinoma cells.The iron deficiency model of C57 mice was successfully constructed by detection of iron content in liver and serum,transferrin saturation in serum,and immunofluorescence of ferritin in liver and lung.H22 cells were labeled with firefly-luciferase?H22-fluc?and cultured to a sufficient number for implantation in situ in C57 mice liver.It was found that the iron deficiency group showed obvious enhancement of metastasis compared with normal group,which suggested that iron deficiency facilitated metastasis of H22 cells in vivo.it was found that the tail intravenous injected H22-fluc cells formed tumors mainly in the lung,and the areas of tumors in iron deficiency group was significantly larger than that in the normal group.5.TFRC knockdown in H22 cells caused intracellular iron deficiency and promoted its metastasis in C57 mice.Detection of TFRC mRNA expression and TfR1 protein expression displayed successful knockdown of TFRC in H22-fluc cells and the content of iron in knockdown cell was lower than that in wildtype cells.H22-fluc-TFRC^knockdown and H22-fluc were implanted in the liver of C57 mice with the same amounts of cells.The results showed that the metastasis of H22-fluc-TFRC^knockdownnockdown cells was significantly enhanced and the pulmonary metastasis was significant compared to H22-fluc cells in C57 mice.Similarly,the metastasis of H22-fluc-TFRC^knockdown cells in C57 mice was increased than H22-fluc cells,which meant intracellular iron deficiency could promote the metastasis of H22 cells in vivo.6.Knockout of TFRC in C57 mice caused iron deficiency and promoted metastasis of H22 cells in vivo.we constructed TFRC knockout C57 mice as genetic model of iron deficiency.Because TFRC homozygous was fatal and TFRC heterozygosity had an appearance of iron deficiency,we used TFRC^-/+mice for following experiments.H22-fluc cells were planted in the liver of TFRC^-/+mice and littermate TFRC^+/+mice.The results showed that H22-fluc cells exhibited significant metastasis and increased lung metastasis in the TFRC^-/+mice.III.Dual-role of SPNS2 in iron deficiency promoting metastasis of HCC cells.1.Iron deficiency increased SPNS2 expression.The results showed that SPNS2 expression in tumor tissues was significantly higher than that in adjacent tumor tissues,suggesting that iron deficiency increased SPNS2expression.We then used DFO and Dp44mT to cause iron deficiency in human primary hepatocytes and mouse primary hepatocytes.The expression of TFRC,HAMP and ferritin showed intracellular iron deficiency,while SPNS2 expression increased significantly.We used three types of human liver cancer cells,Huh7,HepG2 and SMMC-7721,and mouse liver cancer cells H22 to observe effects of iron deficiency on SPNS2 expression.The expression of TFRC,HAMP and ferritin suggested that iron was deficient in cells and SPNS2expression was significantly elevated in all kinds of HCC cells.In nude mice,the liver in iron-deficient group showed a significant increase of SPNS2expression compared to normal group.At the same time,the expression of SPNS2 in the transplanted tumor tissues was higher in iron-deficient group than in normal group.Similarity,the liver in iron-deficient group showed a significant increase of SPNS2expression compared to normal group in C57 mice and the expression of SPNS2 in the transplanted tumor tissues was higher in iron-deficient group than in normal group.2.Upregulation of SPNS2 by iron deficiency modulated immune function.It was found that in the liver tissue and lung tissue,the CD8+T cells and CD69+lymphocyte cells decreased in iron deficient group compared with normal group,as well as in tumor tissues in the liver and lung.Further,we used flow cytometry to detect the change in the percentage of lymphocyte subsets in white cells.The result was that,compared to the normal group,the effector CD8T cells was decreased in liver and lung of iron deficiency group.Especially in lung,B cells and T cells were reduced significantly.3.Silence of SPNS2 expression inhibited the effects of iron deficiency on migration and invasion of HCC cellsInterference of SPNS2 expression inhibited migration and invasion promoted by iron deficiency in Huh7,HepG2 and SMMC-7721,indicating that SPNS2 expression induced by iron deficiency could directly affect migration and invasion of HCC cells in addition to modulating immune function.4.Iron deficiency directly promoted metastasis of HCC cells independent of immune systemWe used complete immunodeficiency mice NCG mice as the research object.Two iron deficiency models were induced by feeding iron deficient feeds to NCG mice or knockdown of transferrin receptors.Each iron deficiency model consisted of liver in situ implantation of Huh7 and H22.The results showed that iron deficiency promoted the metastasis of Huh7and H22 in NCG mice,with significant pulmonary metastasis.In addition to affecting the function of the immune system,the upregulation of SPNS2 induced by iron deficiency can directly affect the metastasis of HCC cells.5.SPNS2 knockout inhibited the promotion effects of iron deficiency on metastasis of HCC cells in vivo.To observe whether iron deficiency promoting the metastasis of HCC cells by upregulating SPNS2 in vivo,we constructed SPNS2 knockout mice.H22-fluc cells were planted in the liver in situ and in the tail vein respectively in the SPNS2^-/-mice and the same nest SPNS2^+/+mice with iron deficient diet.The results showed that the diffusion area and pulmonary metastasis of H22-fluc cells were significantly reduced in SPNS2^-/-mice compared to SPNS2^+/+mice.IV.Iron deficiency upregulated SPNS2 expression by transcription activation of HIF1?and SP1.1.Iron deficiency increased transcriptional activity of HIF1?and SP1 to SPNS2To explore the mechanism of SPNS2 upregulation by iron deficiency,we used transcription factor activity chip to detect changes of activity of 345 transcription factors in the condition of iron deficiency.It was found that the activity of 16 transcription factors changed over twice times?>2 or<0.5?,3 of them were found to have binding sites with SPNS2 promoter by using transcription factors combined sequence database analysis,respectively HIF1?,SP1 and YY1.The ChIP results showed that the transcription activity of HIF1?and SP1 to SPNS2 increased significantly under the condition of iron deficiency,while YY1 had no change,suggesting that HIF1?and SP1 may be the reason for increase of SPNS2 caused by iron deficiency.2.Inhibition of HIF1?or SP1 suppressed the increase of SPNS2 caused by iron deficiency.We used PX478,a widely used HIF1?inhibitors,in Huh7,HepG2 and SMMC-7721 at the same time of DFO intervention.Addition of PX478 significantly suppressed HIF1?expression and the increase of SPNS2 expression caused by DFO,which hinted that iron deficiency upregulated SPNS2 expression through HIF1?.Meanwhile,Addition of Mithramycin A,an inhibitor of SP1,also significantly suppressed the increase of SPNS2expression caused by DFO,indicating that SP1 played a role in SPNS2 upregulation too.ConclusionWe could draw following conclusions from our experimental results:1.Iron deficiency promoted metastasis of HCC cells;2.Iron deficiency promoted metastasis of HCC cells via upregulating SPNS2;3.HIF1?and SP1 played important roles in upregulating SPNS2 expression induced by iron deficiency.