Research On The Abnormal Alterations And Mechanism Of Iron Metabolism In Lung Cancer And Its Influence Of Establishment Of Xenotransplanted Tumor Model

Lung cancer is a kind of malignant tumor with an ever-increasingly high incidence rate and death rate. Especially in the past 50 years, lung cancer has been becoming the leading attack rate among various kinds of cancers, and it tends to happen in younger and female persons. Lung cancer is now the leading death reason among all cancers. Lung cancer, also called bronchiogenic cancer, is malignant cancer originated from bronchus epithelium mucosae, and there are two kinds of lung cancers, namely non-small cell lung cancer (85%) and small cell lung cancer (15%); and the former has three subtypes:prickle cell carcinoma, malignant adenoma and megacell cancer. Based on the research, smoking is the generally believed cause of lung cancer, however, there are still 25% cancers could not be ascribed to smoking, and the death toll is more than three hundred thousand per year. Although there is an unceasing progress in the early non-small cell lung cancer diagnosis criteria and therapy measures, the present situation on this problem is still not enough, and the prognosis is still not good. The present therapy measures are mainly operation, chems and radiotherapy, but the major cases of non-small cell lung cancers are insensitive to the radiotherapy, and the 5 year survival rate still is still lower, and the prevention of it still need new measures. Therefore, its diagnosis means, therapeutic strategy and prevention measures are still the important problem which puzzles us the most.Genesis and development of malignant tumor is resulted in the excess proliferation and deficient apoptosis. All elements involving cell metabolism, proliferation, and apoptosis relate to the genesis, development and infestation and aversion of lung cancer. Iron is one of the essential elements to human body, which plays a decisive role in the cell normal growth and maintaining of its normal functions. As a kind of transition element, iron is prothetic group of nucleinic acid ribonucleotide reductase which synthesizes DNA rate-limiting enzyme, and it participates the DNA synthesis, electron transport, and other important biotic metabolism processes. What's more, mammal hemoglobin mediated transportation of oxygen and scavenger enzyme in many kinds of enzymatic active both relate to the iron participation and its important effects. Iron can reduce the oxygen production when chondriosome creates ATP, and plays important role in the energy transferring. Meanwhile, iron also bears important effects in the maintaining of normal immune function. And, iron is main antileptic of free radicle in many biochemistry systems.So far, the abnormality has been found in many kinds of diseases, such as hepatitis, diabetes, Parkinson's syndrome and endometriosis, which usually demonstrated through the changes of various iron metabolism participation markers. Many cancer patients suffer iron metabolism abnormality, which could be seen through the abnormality of the serological index contents:(1) Fe protein, especially the serological Fn level increased in many kinds of tumors, moreover, in neuroblastoma, serological Fn can exert the function of autocrine growth factor, and have relevance to tumor staging, so as to suggest the function of prognosis; (2) transferring, Tf transports iron from peripheral blood into the cell, and could work as cell growth factor. Actually, some tumor tissues, regardless of vivo or vitro cells, can synthesize siderophilin by themselves to promote cell proliferation and differentiation; (3) Transferrin receptor, so far two kinds of receptors has been identified:TfR1 and TfR2. Related research show that TfR1 expression increase in many kinds of tumors, say brain tumor, and its monoclonal antibody can inhibit the growth of vitro tumor cells;(4) Iron regulatory protein, IRP, through the level after transfection, regulates the expression of Fe protein and transferring, and further to control cells'metabolism. There are two kinds of IRP:IRP1 and IRP2, and vitro experiments indicate both of them can identify and combine their special target RNA series respectively, furthermore, IRP2 are much more sensitive to IRE structure's natural mutation than IRP1. IRP2 works as primary role in the regulating of iron metabolism in some cultured cells such as mouse macrophage J774. Even in some circumstances, there is only IRP2 expression, which indicates that it can work as the only regulator in the iron balance in cells.Research on the relation between iron and lung cancer has been rarely done, and most of the methods are focusing on detection of serum iron protein contents and iron related gene products' expression in tissue, which reveals that the iron metabolism genetic products'expressions in cancer tissues or patients serum are different with the control and innocence diseases, but the genesis cause and the mechanism of iron metabolism in the genesis of lung cancer have not been reported. This study was planning to do on the three level- tissue, cell, and animal, and to research on the following contents:(1) collecting the tissue samples from those first time visit's nonsmall-cell lung cancer and normal latero-cancer tissue, and analyzing the expressions of iron metabolism markers-Fn, Tf, TfR and IRP2 on the levels of gene and protein, and finding out whether there are iron metabolism abnormality in the tissue of nonsmall-cell lung cancer; (2) vitro cultivating A549 cells, and observing cells'proliferation under different levels of FeCl3 and Fe chelator DFO at different points of time; so as to study iron influence on the vitro growth of lung cancer cells and analyze the Fe markers gene and protein expressions at the different conditions;(3) furthermore, using A549 cells to establish the athymic mouse transplantation tumor models after the treatment of different levels of chalybeate and iron chelator, so as to study the iron influence on the animal vivo tumor genesis, and further to discuss the relationship between iron and the genesis of nonsmall-cell lung cancer.This research was doing on the molecular, genetic protein and vivo levels. Immnunohistochemistry, western-blot, PCR, RT-PCR, MTT, Tune, agarose gel electrophoresis and antisense oligonucleotides technology were employed to establish xenogeneic transplanted tumor, and further to explore the expressions of iron metabolism related genes and proteins in lung cancer tissues and vitro cultivating lung cancer cells, and the influence of different iron levels on vitro lung cancer cells. Meanwhile, animal experiments have been performed to find out the iron influence on animal tumor genesis at different levels, furthermore, antisense oligonucleotides technology was applied to make the IRP2 gene silent, and the silent gene's influence on related genes were also investigated, so as to discuss the mechanism of iron metabolism in the genesis of lung cancer systematically with the hope to offer some theoretical basis for lung adenocarcinoma's molecular targeted therapy.Section 1 The expression of iron metabolism relating genes and protein in tissue of lung adenocarcinomaObjectiveTo research whether abnormal iron metabolisms relating genes and expression products exist in lung cancer tissues, so as to probe into the mechanism of iron metabolisms in lung carcinogenesis.Method1. Immunohistochemistry method was employed to detect the protein expression products of iron metabolism relating gene Tf, TfR,Fn, IRP2 in lung cancer tissues.2. RT-PCR method was adopted to detect the gene expression of iron metabolism relating index Tf, TfR,Fn, IRP2.Results1. the positive expression rate of gene Tf in NSCLC tissue is 73.33%, and in Para cancer tissue, the positive expression rate is 23.33%, the differences between the two data has statistical significance (χ2=15.02, P<0.05); the positive expression rate of gene TfR in NSCLC tissue is 63.33%, and in Para cancer tissue, the positive expression rate is 13.33%, the differences between the two data has statistical significance (χ2=15.86, P<0.05); the positive expression rate of gene Fn in lung cancer tissue is 16.66%, and in Para cancer tissue, the positive expression rate is 70.00%, the differences between the two data has statistical significance (χ2=17.38, P<0.05);the positive expression rate of gene IRP2 in NSCLC tissue is 70.00%, and in Para cancer tissue, the positive expression rate is 26.66%, the differences between the two data has statistical significance (χ2=11.28, P<0.05);2. In lung cancer, Tf mRNA is 0.291±0.066, and in Para cancer tissue, the value is 0.175±0.004, the differences between the two data has statistical significance (t=-5.400, P<0.05);In lung cancer, TfR mRNA is 0.441±0.031, and in Para cancer tissue, the value is 0.251±0.028, the differences between the two data has statistical significance (t= 13.198, P<0.05);In lung cancer, Fn mRNA is 0.291±0.019, and in Para cancer tissue, the value is 0.967±0.063, the differences between the two data has statistical significance (t= 30.862, P<0.05);In lung cancer, IRP2 mRNA is 0.275±0.022, and in Para cancer tissue, the value is 0.132±0.011, the differences between the two data has statistical significance (t= 14.992, P<0.05).SummaryThe expression of Fn lowered, while the expression of Tf, TfR and IRP2 upgraded, which shows that the need of iron in the genesis and development of lung cancer increases, and the increasing of iron utilization relies upon the transportation of Tf-TfR, and IRP2 works in the process with its modulating function.Section 2 on the molecular mechanism of iron metabolism in the lung adenocarcinoma carcinogenesisObjectiveTo research the influence of silencing IRP2 gene to the expression of iron metabolism relating genes and their productsMethods1. Cell cultureLung adenocarcinoma A549 cells was cultured in 10% FCS 1640 culture fluid under 37℃with 5%CO2, and the fluid changed regularly while gene transferring.2. Transfection of A549 cells through IRP2 ASODNExperiments were divided into 3 groups:group A:control group (with 20μl/ml level lipoplast);group B:ASODN group;group C:SCODN group;3. Detecting the mRNA expressions of the iron metabolism relating genes Tf, TfR and Fn by RT-PCR technology4. The expressions of iron regulation related genes of IRP2, Tf, TfR and Fn were detected through Western-BlotResults1 The morphological changes from ASODN transfectionThe cells of ASODN transfection group showed changes such as irregular morphological changes, karyopyknosis, nucleus fragmentation, parted chromoplasm, few splitting cells, and the formation of apoptotic body. But the cell shapes from control group and SCODN group are normal on the whole, and displayed good adherence growth.2 the AP:PA transfection expression changes of Tf, TfR and Fn mRNA①Tf mRNA:control group, SCODN group and ASODN group:0.440±0.049, 0.418±0.040,0.468±0.052, and the expression differences among the three groups are not statistically significant (F=2.18, P>0.05);②TfR mRNA:control group, SCODN group and ASODN group:0.791±0.117, 0.856±0.075,0.586±0.061, and the expression differences among the three groups have statistical significance (F=25.671, P<0.05); and, respectively, when compared ASODN group with control and SCODN groups, TfR gene expression from ASODN group degraded significantly (P<0.05), and control group and SCODN group showed no significant difference;③Fn mRNA:control group, SCODN group and ASODN group:0.485±0.038, 0.483±0.038,0.693±0.077, and F check showed the expression differences among the three groups have statistical significance (F=49.748, P<0.05); and, respectively, when compared ASODN group with control and SCODN groups, Fn mRNA gene expression showed higher than those from control and ASODN groups, and statistically significant (P<0.05), and control group and SCODN group showed no significant difference;3 the AP:PA transfection protein expressions of IRP2,Tf, TfR and Fn①Protein expression of Tf: control group, SCODN group and ASODN group are respectively: 0.651±0.052,0.630±0.060,0.593±0.057, and the expression differences among the three groups are not statistically significant (F=2.668,P>0.05); and, respectively, when compared ASODN group with control, Tf protein expression observed statistical significance(P<0.05), but which did not show between ASODN group and SCODN group(P>0.05);②Protein expression of TfR:control group, SCODN group and ASODN group are respectively:0.791±0.117,0.856±0.075,0.586±0.061, and the expression differences among the three groups are statistically significant (F=25.671, P<0.05); and, respectively, when compared ASODN group with control and SCODN groups, TfR protein expression observed statistical significance(P<0.05), but which did not show between ASODN group and SCODN group (P>0.05);③Protein expression of Fn:control group, SCODN group and ASODN group are respectively:0.485±0.038,0.483±0.038,0.693±0.077, and the expression differences among the three groups are statistically significant (F=49.748, P<0.05);and, respectively, when compared ASODN group with control and SCODN groups, Fn protein expression observed statistical significance(P<0.05), but between control and SCODN groups, the protein expression differences have no statistical significance (P>0.05)(?)Protein expression of IRP2:control group, SCODN group and ASODN group are respectively: 0.617±0.100,0.553±0.094,0.120±0.024, and the expression differences among the three groups have statistical significance (F=113.224, P<0.05); when compared ASODN group with control and SCODN groups, the protein expression differences have statistical significance (P<0.05).Summary1 After IRP2 ASODN transfection, IRP2 protein's expression of A549 cells lowered significantly;2 After IRP2 ASODN transfection, TfR mRNA and protein expression lowered; Fn mRNA and protein expression increased; and TfmRNA and protein expression remain unchanged.3 In lung cancer, IRP2 may regulate the expression of TfR and Fn through the quantity of proteins, and further to modulate iron metabolism.Section 3 the influences on proliferation and apoptosis of A549 cell strains under different iron conditionsObjectiveTo study the influence of adding and removing iron on proliferation and apoptosis process, iron metabolism related genes and their expression products, and further to discuss the contribution and mechanism of iron metabolism in lung cancer genesis at cellular level.Methods1 experiment grouping:①blank control group;②50μmol/L FeCl3 group;③100μmol/L FeCl3 group;④50μmol/L deferoxamine (DFO) group;⑤100μmol/L deferoxamine (DFO) group.2 vitro reproductive activity detection through MTT method3 apoptosis information observation through flow cytometry4 RT-PCR detection about mRNA levels of the iron metabolism related index-Fn, Tf, TfR, and IRP2 from every group5 Western blot detection about protein expression of the iron metabolism related index-Fn, Tf, TfR, and IRP2 from every groupResults1. FeCl3 influence on vitro proliferation of vitro cultured A549 cells1.1 proliferation effect of FeCl3 on A549 cells When FeCl3-50 and FeCl3-100 treatment on A549 cells for 12h, the proliferation rates are respectively: 0.242±0.02,0.328±0.027, and the difference between the two data showed statistical significance (t=-8.128, P<0.05); for 24h, the rates are respectively:0.698±0.066,0.943±0.071, and the difference between the two data showed statistical significance (t=-7.997, P<0.05); for 48h, the rates are respectively: 0.843±0.082,1.113±0.094, and the difference between the two data showed statistical significance (7=-6.842, P<0.05).Based on the time gradient, after the FeCl3-50 and FeCl3-100 treatment for 12h and 24h, the proliferation rates gradually increased with the lasting of time (P<0.05), but the 48h treatment values bear no statistical significance with that of 24h (P>0.05).1.2 DFO influence on vitro proliferation of vitro cultured A549 cellsWhen DFO-50及DFO-100 treatment on A549 cells for 12h, the proliferation rates are respectively:0.866±0.011,0.819±0.016, and the difference between the two data showed statistical significance (f=7.56100, P<0.05); for 24h, the rates are respectively: 0.608±0.03,0.461±0.053, and the difference between the two data showed statistical significance(t=1.62700, P<0.05); for 48h, the rates are respectively: 0.59±0.043,0.38±0.055, and the difference between the two data showed statistical significance (7=9.58700, P<0.05).Based on the time gradient, after the DFO-50 and DFO-100 treatment for 12h and 24h, the proliferation rates gradually increased with the lasting of time (P<0.05), but the 48h treatment values bear no statistical significance with that of 24h (P>0.05).2. RT-PCR detection on the mRNA levels of Tf, TfR,Fn, IRP2 after FeCl3 and DFO treatment on A549 cells2.1 FeCl3 influence on the Tf mRNA level of A549 cellsTf mRNA levels of A549 cells after 12h FeCl3 treatment: control group, FeCl3-50 group and FeCl3-100 group are respectively: 0.423±0.043,0.396±0.027, 0.344±0.02, and the mRNA levels among the three groups have significant difference (F= 16.413, P<0.05);Tf mRNA levels of A549 cells after 24h FeCl3 treatment: control group, FeCl3-50 group and FeCl3-100 group are respectively: 0.435±0.039,0.333±0.044, 0.265±0.023, and the mRNA levels among the three groups have significant difference (F=55.099, P<0.05).Based on the time gradient, FeCl3-50 group's Tf mRNA levels of 12h and 24h gradually lowered with the lasting of time, and the mRNA levels differences have statistical significance (P<0.05); FeCl3-100 group's Tf mRNA levels of 12h and 24h gradually lowered with the lasting of time, and the mRNA levels differences have statistical significance (P<0.05).2.2 FeCl3 influence on the TfR mRNA level of A549 cellsTfR mRNA levels of A549 cells after 12h FeCl3 treatment:control group, FeCl3-50 group and FeCl3-100 group are respectively:0.479±0.045,0.469±0.039, 0.385±0.0322, and the mRNA levels among the three groups have significant difference (F=17.576, P<0.05).TfR mRNA levels of A549 cells after 24h FeCl3 treatment:control group, FeCl3-50 group and FeCl3-100 group are respectively:0.464±0.045,0.360±0.031, 0.321±0.010, and the TfR mRNA levels among the three groups have significant difference (F=52.702, P<0.05).Based on the time gradient, FeCl3-50 group's TfR mRNA levels of 12h and 24h gradually increased with the lasting of time, and the mRNA levels differences have statistical significance (P<0.05); FeCl3-100 group's TfR mRNA levels of 12h and 24h gradually increased with the lasting of time, and the mRNA levels differences have statistical significance (P<0.05).2.3 FeCl3 influence on the Fn mRNA level of A549 cellsFn mRNA levels of A549 cells after 12h FeCl3 treatment: control group, FeCl3-50 group and FeCl3-100 group are respectively: 0.380±0.032,0.460±0.046, 0.599±0.047, and the mRNA levels among the three groups have statistical significance (F=69.437, P<0.05);Fn mRNA levels of A549 cells after 24h FeCl3 treatment: control group, FeCl3-50 group and FeCl3-100 group are respectively: 0.379±0.019,0.501±0.050, 0.851±0.054, and the mRNA levels among the three groups have statistical significance (F=316.410, P<0.05).Based on the time gradient, FeCl3-50 group's Fn mRNA levels of 12h and 24h gradually increased with the lasting of time, and the differences have statistical significance (P<0.05) FeCl3-100 group's Fn mRNA levels of 12h and 24h gradually increased with the lasting of time, and the mRNA levels differences have statistical significance (P<0.05).2.4 FeCl3 influence on the IRP2 mRNA level of A549 cellsIRP2 mRNA levels of A549 cells after 12h FeCl3 treatment: control group, FeCl3-50 group and FeCl3-100 group are respectively: 0.304±0.021,0.287±0.020, 0.205±0.020, and the IRP2 mRNA levels among the three groups have significant difference (F=68.144, P<0.05).IRP2 mRNA levels of A549 cells after 24h FeCl3 treatment: control group, FeCl3-50 group and FeCl3-100 group are respectively: 0.311±0.022,0.256±0.022, 0.206±0.011, and the IRP2 mRNA levels among the three groups have significant difference (F=76.749, P<0.05).Based on the time gradient, FeCl3-50 group's IRP2 mRNA levels of 12h and 24h gradually increased with the lasting of time (P<0.05), and the mRNA levels differences have statistical significance (P0.05).2.5 DFO influence on the Tf mRNA level of A549 cellsTf mRNA levels of A549 cells after 12h DFO treatment:control group, DFO-50 group and DFO-100 group are respectively: 0.423±0.043,0.483±0.042,0.535±0.029, and the mRNA levels among the three groups have significant difference (F=21.134, P<0.05).Tf mRNA levels of A549 cells after 24h DFO treatment:control group, DFO-50 group and DFO-100 group are respectively: 0.435±0.039,0.516±0.058,0.555±0.062, and the mRNA levels among the three groups have significant difference (F=l2.987, P<0.05).Based on the time gradient, DFO-50 group's Tf mRNA levels of 12h and 24h gradually increased with the lasting of time (F<0.05), and the mRNA levels differences, have statistical significance (P<0.05);DFO-100 group's Tf mRNA levels of 12h and 24h gradually increased with the lasting of time, and the mRNA levels differences have statistical significance (P<0.05).2.6 DFO influence on the TfR mRNA level of A549 cellsTf mRNA levels of A549 cells after 12h DFO treatment: control group, DFO-50 group and DFO-100 group are respectively:0.479±0.045,0.484±0.043,0.597±0.067, and the mRNA levels among the three groups have significant difference (F=15.888, P<0.05).Tf mRNA levels of A549 cells after 24h DFO treatment: control group, DFO-50 group and DFO-100 group are respectively: 0.464±0.045,0.521±0.039,0.652±0.049, and the mRNA levels among the three groups have significant difference (F=46.822, P<0.05). Based on the time gradient, DFO-50 group's TfR mRNA levels of 12h and 24h gradually increased with the lasting of time (P<0.05), and the mRNA levels differences have statistical significance (F=316.410, P<0.05);DFO-100 group's TfR mRNA levels of 12h and 24h gradually increased with the lasting of time, and the mRNA levels differences have statistical significance (P<0.05).2.7 DFO influence on the Fn mRNA level of A549 cellsFn mRNA levels of A549 cells after 12h DFO treatment:control group, DFO-50 group and DFO-100 group are respectively: 0.380±0.032,0.292±0.015,0.23±0.021, and the mRNA levels among the three groups have significant difference (F=98.313, P<0.05);Fn mRNA levels of A549 cells after 12h DFO treatment:control group, DFO-50 group and DFO-100 group are respectively: 0.379±0.019,0.215±0.024,0.196±0.019, and the mRNA levels among the three groups have significant difference (F=235.643, P<0.05).Based on the time gradient, FeC13-50 group's Fn mRNA levels of 12h and 24h gradually lowered with the lasting of time, and the mRNA levels differences have statistical significance (F=316.410, P<0.05); FeCl3-100 group's Fn mRNA levels of 12h and 24h gradually lowered with the lasting of time, and the mRNA levels differences have statistical significance (P<0.05).2.8 DFO influence on the IRP2 mRNA level of A549 cellsIRP2 mRNA levels of A549 cells after 12h DFO treatment: control group, DFO-50 group and DFO-100 group are respectively: 0.304±0.021,0.412±0.024, 0.445±0.063, and the IRP2 mRNA levels among the three groups have significant difference (F=33.015, P<0.05).IRP2 mRNA levels of A549 cells after 24h DFO treatment: control group, DFO-50 group and DFO-100 group are respectively: 0.311±0.022,0.401±0.019, 0.541±0.051, and the IRP2 mRNA levels among the three groups have significant difference (F=117.266, P<0.05)Based on the time gradient, DFO-50 group's IRP2 mRNA levels of 12h and 24h gradually lowered with the lasting of time (P<0.05), and the mRNA levels differences have no statistical significance (F=316.410, P<0.05); DFO-100 group's IRP2 mRNA levels of 12h and 24h gradually increased with the lasting of time, and the mRNA levels differences have statistical significance (P<0.05).3 Western-blot detection of the Tf, TfR,Fn, and IRP2 protein levels of A549 cells after FeCl3 and DFO treatment3.1 FeCl3 influence on the Tf protein level of A549 cellsTf protein levels of A549 cells after 12h FeCl3 treatment:control group, FeCl3-50 group and FeCl3-100 group are respectively: 0640±0.023,0.607±0.030, 0.574±0.035, and the Tf protein levels among the three groups have significant difference (F=11.462, P<0.05)Tf protein levels of A549 cells after 24h FeCl3 treatment:control group, FeCl3-50 group and FeCl3-100 group are respectively: 0.626±0.044,0.567±0.036, 0.512±0.032, and the Tf protein levels among the three groups have significant difference (F=22.810, P<0.05)Based on the time gradient, FeCl3-50 group's Tf protein levels of 12h and 24h gradually lowered with the lasting of time (P<0.05), and the differences have statistical significance (P<0.05); FeCl3-100 group's Tf protein levels of 12h and 24h gradually lowered with the lasting of time, and the differences have statistical significance (P<0.05).3.2 FeCl3 influence on the TfR protein level of A549 cellsTfR protein levels of A549 cells after 12h FeCl3 treatment:control group, FeCl3-50 group and FeCl3-100 group are respectively: 0.762±0.033,0.643±0.022, 0.426±0.034, and the TfR protein levels among the three groups have significant difference (F=316.051, P<0.05).TfR protein levels of A549 cells after 24h FeCl3 treatment:control group, FeCl3-50 group and FeCl3-100 group are respectively: 0.793±0.051,0.570±0.034, 0.341±0.025, and the.TfR protein levels among the three groups have significant difference (F=352.725, P<0.05).Based on the time gradient, FeCl3-50 group's TfR protein levels of 12h and 24h gradually lowered with the lasting of time, and the differences have statistical significance (P<0.05); FeCl3-100 group's TfR protein levels of 12h and 24h gradually lowered with the lasting of time, and the differences have statistical significance (P<0.05).3.3 FeCl3 influence on the Fn protein level of A549 cellsFn protein levels of A549 cells after 12h FeCl3 treatment:control group, FeCl3-50 group and FeCl3-100 group are respectively: 0.497±0.038,0.507±0.020, 0.550±0.050, and the protein levels among the three groups have significant difference (F=5.528,P<0.05). Fn protein levels of A549 cells after 24h FeCl3 treatment:control group, FeCl3-50 group and FeCl3-100 group are respectively: 0.471±0.029,0.647±0.051, 0.902±0.09, and the Fn protein levels among the three groups have significant difference (F=121.661, P<0.05).Based on the time gradient, FeCl3-50 group's Fn protein levels of 12h and 24h gradually increased with the lasting of time (P<0.05), and the differences have statistical significance (P<0.05); FeCl3-100 group's Fn protein levels of 12h and 24h gradually increased with the lasting of time, and the differences have statistical significance (P<0.05).3.4 FeCl3 influence on the IRP2 protein level of A549 cellsIRJP2 protein levels of A549 cells after 12h FeCl3 treatment: control group, FeCl3-50 group and FeCl3-100 group are respectively:0.589±0.033,0.313±0.021, 0.303±0.025, and the IRP2 protein levels among the three groups have significant difference (F=364.328, P<0.05)IRP2 protein levels of A549 cells after 24h FeCl3 treatment:control group, FeCl3-50 group and FeCl3-100 group are respectively: 0.589±0.024,0.277±0.032, 0.299±0.220, and the IRP2 protein levels among the three groups have significant difference (F=18.185, P<0.05)Based on the time gradient, FeCl3-50 group's IRP2 protein levels of 12h and 24h gradually lowered with the lasting of time, and the differences have statistical significance (P<0.05); FeCl3-100 group's IRP2 protein levels of 12h and 24h gradually lowered with the lasting of time, and the differences have no statistical significance (P>0.05).3.5 DFO influence on the Tf protein level of A549 cellsTf protein levels of A549 cells after 12h DFO treatment: control group, DFO-50 group and DFO-100 group are respectively: 0.574±0.035,0.826±0.121,0.822±0.0816, and the Tf protein levels among the three groups have significant difference (F=27.946,P<0.05)Tf protein levels of A549 cells after 24h DFO treatment: control group, DFO-50 group and DFO-100 group are respectively: 0.626±0.044,0.734±0.058,0.796±0.063, and the Tf protein levels among the three groups have significant difference (F=23.917,P<0.05)Based on the time gradient, DFO-50 group's Fn protein levels of 12h and 24h gradually lowered with the lasting of time, and the levels differences have statistical significance (P>0.05); DFO-100 group's Fn protein levels of 12h and 24h gradually increased with the lasting of time, and the levels differences have no statistical significance (P>0.05).3.6 DFO influence on the TfR protein level of A549 cellsTfR protein levels of A549 cells after 12h DFO treatment:control group, DFO-50 group and DFO-100 group are respectively: 0.762±0.033,0.724±0.077, 0.945±0.079, and the TfR protein levels among the three groups have significant difference (F=31.480, P<0.05).TfR protein levels of A549 cells after 24h DFO treatment: control group, DFO-50 group and DFO-100 group are respectively: 0.793±0.051,0.623±0V069, 0.938±0.077, and the TfR protein levels among the three groups have significant difference (F=55.869, P<0.05).Based on the time gradient, DFO-50 group's TfR protein levels of 12h and 24h gradually lowered with the lasting of time, and the levels differences have statistical significance (P<0.05); DFO-100 group's TfR protein levels of 12h and 24h gradually lowered with the lasting of time, and the levels differences have no statistical significance (P>0.05).3.7 DFO influence on the Fn protein level of A549 cellsFn protein levels of A549 cells after 12h DFO treatment:control group, DFO-50 group and DFO-100 group are respectively:0.497±0.038, O.38O±0.O39,0.319±0.036, and the Fn protein levels among the three groups have significant difference (F=57.663, P<0.05)Fn protein levels of A549 cells after 24h DFO treatment:control group, DFO-50 group and DFO-100 group are respectively: 0.471±0.029,0.343±0.020,0.168±0.017, and the protein levels among the three groups have significant difference (F=456.794, P<0.05)Based on the time gradient, DFO-50 group's Fn protein levels of 12h and 24h gradually lowered with the lasting of time, and the levels differences have no statistical significance (P>0.05);DFO-100 group's Fn protein levels of 12h and 24h gradually increased with the lasting of time, and the levels differences have statistical significance (P<0.05).3.8 DFO influence on the IRP2 protein level of A549 cellsIRP2 protein levels of A549 cells after 12h DFO treatment:control group, DFO-50 group and DFO-100 group are respectively: 0.589±0.O33,0.659±0.047, 0.927±0.0689, and the IRP2 protein levels among the three groups have significant difference (F=119.463, P<0.05).IRP2 protein levels of A549 cells after 24h DFO treatment:control group, DFO-50 group and DFO-100 group are respectively: 0.589±0.024,0.725±0.049, 0.954±0.075, and the IRP2 protein levels among the three groups have significant difference (F=118.096, P<0.05)Based on the time gradient, DFO-50 group's IRP2 protein levels of 12h and 24h gradually increased with the lasting of time, and the levels differences have statistical significance (P<0.05); DFO-100 group's IRP2 protein levels of 12h and 24h gradually increased with the lasting of time, and the levels differences have no statistical significance (.P>0.05).4 FeCl3 and DFO influences on A549 cells apoptosis4.1 Morphological observation on A549 cells'apoptosisDifferent levels of FeCl3 and DFO treatment on A549 cells for 24h, typical morphological characteristics of apoptosis cells can be observed through Hematoxylin-eosin (HE) staining: cell shrinkage, weakening refraction feature, cell endoparticle increasing; and more cell apoptosis could be seen in the DFO treatment.4.2 Tunnel detection on cells apoptosisDFO facilitates A549 cells...