Biological Function And Mechanism Of Iron Metabolism-related Gene RRM2 In Hepatocellular Carcinoma Cells

BackgroundPrimary liver cancer is one of the most common malignant tumors worldwide,with complex etiology,insidious onset,and high malignancy.Hepatocellular carcinoma(HCC)accounts for approximately 75-85%of primary liver cancers.HCC is not sensitive to systemic chemotherapy and radiotherapy.Although there have been great developments in surgical and drug treatments in recent decades,patients who are diagnosed for the first time have often entered the middle and advanced stages,and thus miss the optimal treatment time,resulting in poor prognosis for HCC patients.Therefore,it is of great significance to predict the prognosis of patients with HCC and to study the mechanism of HCC occurrence and development.Iron is an essential element for living organisms and participates in various important physiological processes.Studies in the past decades have found that iron has dual properties,which can promote cell growth or cell death.It has been observed in cancer cells that in order to meet the need for rapid proliferation,cancer cells are more dependent on iron and are more susceptible to iron depletion.Because of the massive increase in the demand for iron by cells,iron metabolism disorders are common in different types of cancer,including lung cancer,gastric cancer,breast cancer,colorectal cancer,which are involved in the occurrence,development,invasion and metastasis of tumors.Liver is the main storage and metabolic organ of iron in the human body,which plays an important role in maintaining iron homeostasis.At present,studies have confirmed that iron metabolism disorder is an important risk factor for HCC and is closely related to the occurrence and development of HCC.However,the effect of iron metabolism-related genes on HCC is not fully understood,and there is no relevant research report on the prognosis of HCC patients.Therefore,this study used the comprehensive analysis method of bioinformatics to screen out key prognostic genes,which used to construct a prognostic model,and systematically analyzed the relationship between iron metabolism-related genes and the prognosis of patients with HCC.According to the results of the prognostic genes we screened,combined with a large number of literature readings,it was found that RRM2 gene was remarkably overexpressed in HCC patients,and there were currently few researches on the relationship between RRM2 expression level and HCC.Therefore,we selected RRM2 as the target gene to investigate the effect of RRM2 expression level on the biological functions of HCC and related molecular regulation mechanisms,which helped to further explore the process of HCC occurrence and development.It provided new insights for the targeted drug therapy of HCC patients,which has important clinical significance.Part Ⅰ:Effect of iron metabolism-related genes in the prognosis prediction of hepatocellular carcinomaObjectiveIn the present study,we aimed to seek the prognostic factors of iron metabolism genes closely correlated to the poor prognosis of HCC based on the bioinformatics analyses.Methods1.The RNA sequencing(RNA-seq)data,DNA methylation data and clinical information of HCC patients were extracted from The Cancer Genome Atlas(TCGA)database for the subsequent analysis.Screening out differentially expressed genes(DEGs)through R software.Then,we downloaded iron metabolism-related gene sets from the Molecular Signatures Database(MSigDB),and combined with DEGs to obtain differentially expressed iron metabolism-related genes.2.Using the methylation data of HCC patients,the correlation between the gene expression of DEGs and the methylation of CpG sites was analyzed.3.Perform LASSO Cox regression analysis,univariate Cox regression analysis,and multivariate Cox regression analysis on the iron metabolism-related and methylated genes identified to obtain DEGs closely related to the prognosis of HCC.4.A prognostic model of these gene signatures was constructed to predict the overall survival(OS)of HCC.The risk score was calculated as formula,and the patients in the TCGA of HCC were ranked into high-risk and low-risk groups according to the median value of risk score.The Kaplan-Meier(K-M)survival and Receiver Operating Characteristic(ROC)curve analysis were further performed to evaluate the prognostic significance of gene signature.The risk score and clinical information of HCC patients were subjected to a multivariate Cox analysis to determine whether the risk score of the prognostic model can be used as an independent prognostic factor for HCC.5.We further demonstrated the reliability of the prognostic model from an independent HCC cohort from the International Cancer Genome Consortium(ICGC).6.Weighted Gene Co-expression Network Analysis(WGCNA)was performed to construct the co-expression network based on the gene signature and synergistic gene modules.In addition,the Gene Set Enrichment Analysis(GSEA)identified the enrichment pathways related to the gene signatures in HCC.7.We harvested cancerous and adjacent non-cancerous specimens from 12 HCC patients who underwent surgery in the Shandong Provincial Hospital from September to October 2020.Use immunohistochemical staining to identify the expression of target genes in HCC tissues.Results1.We identified 71 DEGs related to iron metabolism between the cancerous and non-cancerous groups,of which 29 were upregulated and 42 were downregulated from TCGA.Performed a correlation analysis between the expression level and CpG site methylation of the 71 co-differentially expressed genes in HCC,then used a LASSO regression analysis,univariate Cox regression and multivariate Cox regression analysis on the differentially expressed genes respectively.We identified 4 iron metabolism-related genes related to the prognosis of HCC,including RRM2,FTCD,CYP2C9 and ATP6V1C1.2.A prognostic model was constructed based on the expression levels and the corresponding coefficients of the four genes,and was used to calculate a risk score for patients with HCC from TCGA.The risk score formula was:Risk score=RRM2*0.24+FTCD*(-0.087)+CYP2C9*(-0.097)+ATP6VIC 1*0.27.The K-M analysis revealed that the survival rates of patients in high-risk group were significantly lower compared with the patients in low-risk group(p=3.356e-06).Furthermore,time-dependent ROC curve showed that the area under the curve(AUC)was 0.74 at 1 year,0.67 at 3 years,and 0.65 at 5 years.Importantly,the results indicated that the risk score was an independent variable correlated with the prognosis in HCC patients(p=2.8e-06,HR=1.7,95%CI:1.4-2.1).3.We further demonstrated the reliability of the prognostic model in another independent patient cohort from the ICGC.Consistent with the results in the TCGA cohort,the K-M curve showed that patients in high-risk group had shorter survival probability than those in low-risk group(p=4.534e-07).The AUC was 0.77 at 1 year and 3 years.4.A total of 213 target genes was found to be co-expressed with the 4-gene signature in the WGCNA network.In addition,GSEA showed that these iron metabolism-related genes were associated with intracellular phagocytic pathway,MAPK signaling pathway,PI3K-AKT signaling pathway.5.Consistent with our bioinformatics analysis,immunohistochemical results show that the protein levels of RRM2 and ATP6V1C1 were increased and that of FTCD and CYP2C9 were decreased in cancerous tissues compared to that in adjacent non-cancerous tissue in HCC patient.Conclusions1.The present study identified genes related to iron metabolism that are key to HCC prognosis,which were closely associated with poor prognosis in HCC patients.2.We constructed a prognostic model that helped calculate risk scores based on the expression level of four genes.The calculated risk score was an independent predictor of HCC prognosis.Part Ⅱ:The biological function of RRM2 in hepatocellular carcinoma cellsObjectiveIn the present study,we further clarified the expression of RRM2 in the tissues of HCC patients.Explored the effect of the expression level of iron metabolism-related gene RRM2 on the biological functions of HCC cells in vitro and vivo.Methods1.TCGA database was.used to analyze the expression of RRM2 in HCC patients,and the relationship between the expression level of RRM2 and the prognosis of HCC.2.Real-time quantitative PCR(qRT-PCR)and Western blot were used to detect the expression level of RRM2 in HCC tissuels and adjacent non-cancerous tissues.3.Knockdown of RRM2 in MHCC97H and Huh7 cell lines were used for siRNA studies,and BEL7402 cell line was used for RRM2 overexpression studies.qRT-PCR and Western blot assays were performed to detect total RNA and protein expression in the three HCC cell lines after 48 hours transfection.4.The effects of RRM2 knockdown and overexpression on the proliferation of HCC cells were analyzed by MTT assay,cell clone formation assay and 5-ethynyl-2’-deoxyuridine(EdU)proliferation assay.5.Through cell cycle analysis,analyze the impact of RRM2 knockdown and overexpression on the cell cycle.6.Annexin V-FITC/PI staining was used to analyze the effect of RRM2 knockdown and overexpression on the apoptosis of HCC cells.At the same time,the expressions of apoptosis-related proteins Bcl-2,Bax and PARP in the three transfected HCC cell lines were detected by Western blot.7.The effects of RRM2 knockdown and overexpression on migration of HCC cells were analyzed by Transwell assay and Wound-healing assay.At the same time,the expressions of migration-related proteins E-cadherin,N-cadherin and Vimentin in the transfected HCC cells were detected by Western blot.8.In vivo experiments were used to determine the effect of RRM2 expression on the proliferation of HCC.In the MHCC97H cell line,RRM2 was stably knocked down by lentivirus infection to construct a subcutaneous xenograft tumor proliferation model in nude mice.The subcutaneous xenograft tumor growth curve was plotted,and the expression of Ki-67 protein was detected by immunohistochemistry.Cell apoptosis was detected by Tunel assay.Results1.Through the TCGA database,it was found that RRM2 was overexpressed in HCC tissues compared with adjacent non-cancerous tissues from TCGA.The overall survival and disease-free survival of HCC patients with overexpress of RRM2 was significantly shorter than that of patients with low RRM2 expression.2.Compared with matched adjacent tissues,qRT-PCR and Western blot showed that the expression level of RRM2 was significantly upregulated in HCC tissues(p<0.05).3.The qRT-PCR was used to verify the transfection efficiency of the three liver cancer cells,and then the Western blot was used to further verify.qRT-PCR results showed that the expression of RRM2 in the MHCC97H and Huh7 cell lines were significantly downregulated(p<0.001),while the expression of RRM2 in the BEL7402 cell line was significantly upregulated(p<0.001).Western blot assay verified the above results.The RRM2 protein expression of the MHCC97H and Huh7 cell lines were significantly downregulated(p<0.001),while the RRM2 protein expression of the BEL7402 cell line was significantly upregulated(p<0.05).4.The results of MTT assay,clone formation assay and EdU assay all showed that the low expression of RRM2 could inhibit the proliferation of HCC,and the overexpression of RRM2 could promote the proliferation of HCC.The differences were statistically significant.5.The proportion of RRM2 low expressing MHCC97H and Huh7 cells in the G0/G1 phase were increased,while the proportion of S phase were decreased significantly(p<0.05).Conversely,the proportion of RRM2 overexpressing BEL7402 cells in G0/G1 and G2/M phases were decreased,while the proportion of S phase cells were increased significantly(p<0.05).6.Apoptosis assay showed that the apoptosis rate increased significantly in RRM2 low expressing MHCC97H and Huh7 cells(p<0.001).On the contrary,the apoptosis rate decreased significantly in RRM2 overexpressing BEL7402 cells(p<0.01).7.Western blot results showed that in MHCC97H and Huh7 cell lines,downregulated RRM2 expression significantly increased the expression levels of Bax and PARP(p<0.001),and the expression of Bcl-2 was decreased(p<0.001).Conversely,increased RRM2 expression significantly decreased the expression levels of Bax and PARP(p<0.001),while increased the expression level of Bcl-2(p<0.001).8.Transwell assay showed that the migration of MHCC97H and Huh7 cells were significantly inhibited(p<0.001).In contrast,the migration of BEL7402 HCC cells was significantly enhanced(p<0.01).Wound-healing assay results showed that knockdown of RRM2 in MHCC97H and Huh7 cells,the migration rate was significantly decreased.Meanwhile,the migration rate of RRM2 overexpressing BEL7402 cells was significantly increased than that of the control group.The differences were statistically significant.9.Western blot results showed that in MHCC97H and HUH7 cell lines,downregulated RRM2 expression resulted in significantly increased E-cadherin expression(p<0.001),while decreased N-cadherin and Vimentin expression,with statistically significant differences.On the contrary,the increased RRM2 expression in Bel-7402 cell line resulted in a significant decrease in E-cadherin expression(p<0.001),while the significant increase in N-cadherin and Vimentin expression(p<0.001).10.Collected lentivirus-infected MHCC97 cells for subcutaneous xenograft tumor proliferation assay in nude mice.The results showed that the volume and weight of subcutaneous xenograft tumors in the sh-RRM2 group that knocked down RRM2 were significantly lower than those in the control group(p<0.05).The results of immunohistochemistry showed that the Ki-67 protein positive staining rate of sh-RRM2 group was significantly lower than that of control group(p<0.05).Tunel assay showed that the apoptosis rate of sh-RRM2 group was significantly higher than that of control group(p<0.05).Conclusions1.RRM2 was remarkable overexpression in HCC patients and was associated with poor prognosis.2.Inhibition of RRM2 expression could inhibit the proliferation and migration of HCC cells,induced cell cycle arrest and promoted cell apoptosis.Overexpression of RRM2 could promote the proliferation and migration of HCC cells,and have a significant inhibitory effect on apoptosis.3.The low expression of RRM2 could inhibit the proliferation of HCC and promote apoptosis in vivo.4.We have conducted a comprehensive analysis of the biological functions of the iron metabolism-related gene RRM2 in HCC cells,providing new insights and directions for targeted drug therapy of HCC.Part Ⅲ:The mechanism of RRM2 promoting the proliferation and migration of hepatocellular carcinoma cellsObjectiveWe studied the relevant molecular mechanisms of RRM2 promoting the proliferation and migration of hepatocellular carcinoma cells.Methods1.Analyzed the signal pathways that may be affected by RRM2 through bioinformatics technology,and found the target signal pathway that needed to be studied.2.Western blot was used to detect changes in the expression of signaling pathway proteins in MHCC97H and Huh7 cell lines that knockdown of RRM2.3.Rescue experiments were conducted to verify whether upregulation of key regulatory proteins in the signaling pathway could reverse the effects of knockdown RRM2 expression on apoptosis and migration of HCC cells.Results1.Using the hallmark gene set of GSEA for enrichment analysis,it was found that RRM2 was related to the activation of mTOR signaling pathway.2.The expression levels of p-Akt,p-mTOR and p-p70S6K were all significantly downregulated in MHCC97H and Huh7 cell lines that knockdown of RRM2,and the differences were statistically significant(p<0.05).3.In MHCC97H and Huh7 cell lines with RRM2 knockdown,plasmids were used to construct Akt overexpression.Apoptosis assay confirmed that upregulation of Akt expression resulted in a significant decrease in the rate of apoptosis(p<0.05),and achieved the expected rescue effect.The results of the Transwell assay showed that upregulation of Akt could significantly increase the migration ability of HCC cells(p<0.05),and the expected rescue effect was also achieved.ConclusionsThe study suggested that RRM2 could promote the occurrence and development of HCC through the Akt/mTOR/p70S6K signaling pathway.