| BackgroundIn China,primary liver cancer is the second leading cause of cancer-related death and ranks fourth in terms of incidence.Hepatocellular carcinoma(HCC)is the most common pathological type of primary liver cancer and the most common malignant tumor of the liver.It has a complex etiology,insidious onset,rapid progression and high malignancy.Most patients are in the middle to late stage at the time of first diagnosis and lose the opportunity of radical surgical resection,therefore,the prognosis is poor.Systemic drug therapy is currently the main treatment for advanced HCC.Sorafenib(SF),a common standard first-line targeted therapy,can significantly prolong the overall survival of intermediate to advanced HCC.However,sorafenib is initially effective in only about 30%of clinical applications and eventually develops acquired drug resistance.Thus,poor sensitivity to sorafenib has been an important factor limiting the efficacy of targeted therapy for advanced HCC.Therefore,studying the molecular mechanism of sorafenib against cancer and exploring the discovery of new therapeutic targets are particularly important for developing antitumor drugs and improving the efficacy of sorafenib in treating HCC.As a new type of programmed cell death,ferroptosis is morphologically,biochemically and genetically completely different from apoptosis,necrosis and autophagy.It was first named by Stockwell et al.in 2012.Ferroptosis is characterized by intracellular iron-dependent accumulation of lipid peroxides and lipid reactive oxygen species(ROS),causing an imbalance in the intracellular redox environment and leading to cell death.Currently,sorafenib has been identified as an inducer of ferroptosis by blocking System Xc-mediated cysteine input and reducing glutathione biosynthesis,leading to intracellular glutathione(GSH)depletion and inducing the accumulation of lipid peroxides leading to cell death.The present study shows that sorafenib induces ferroptosis in HCC not by inhibition of multi-kinase activity but by inhibition of System Xc-as mentioned above,and that sorafenib induces ferroptosis more than the proapoptotic effect that depends on its kinase inhibitory activity.It is clear that ferroptosis plays an important role in sorafenib treatment of HCC.Therefore,more and more researchers are focusing on the discovery of regulatory proteins,small molecule compounds,and pathways involved in the regulation of ferroptosis to improve the effectiveness of cancer drug therapy.N6-methyladenosine(m6A)is the most common internal RNA modification that occurs at the post-transcriptional level in eukaryotes.m6A modifications affect multiple aspects of RNA metabolism,from RNA processing,nuclear export,RNA translation to decay,thereby controlling protein expression in a post-transcriptional manner.Several recent studies have shown that m6A can be involved in fine-tuning programmed cell death pathways including apoptosis,autophagy and ferroptosis,which in turn regulate cancer development and progression.m6A is assembled by m6A methyltransferase(Writer),recognized by reading protein(Reader),and erased by m6A demethylase(Eraser).It has been demonstrated that IGF2BP3,a known reader protein(Reader)for m6A,is able to recognize and bind to the m6A modification site of its target mRNA,thereby regulating the expression level of the target RNA and thus affecting the protein expression level.IGF2BP3 is an insulin-like growth factor-II mRNA binding protein(IGF2BPs,including IGF2BP3 is an important member of the IGF2BP1/2/3)family,and upregulation of IGF2BP3 is closely associated with tumor invasion,early recurrence and poor prognosis in various human cancers,including HCC.In vitro studies have also shown that IGF2BP3 is post-transcriptionally regulated by tumor growth,drug resistance and metastasis.However,to date,the role and molecular mechanisms of IGF2BP3 in ferroptosis of HCC cells are not clear.In this study,the gene expression of IGF2BP3 in HCC and its correlation with HCC prognosis were firstly further explored by mining the public gene expression data.IGF2BP3 was found to be highly expressed in HCC tumors and portal vein tumor thrombus,and high expression was associated with early recurrence and poor prognosis.Then by detecting the levels of reactive oxygen species(ROS),Fe2+,malondialdehyde(MDA)and ferroptosisrelated marker proteins in HCC cells after sorafenib treatment,we found that knockdown of IGF2BP3 significantly enhanced the sensitivity of HCC cells to sorafenib-induced ferroptosis.Furthermore,NRF2 mRNA was identified as an important target of IGF2BP3 by bioinformatics analysis,RNA binding protein immunoprecipitation(RIP)and RNA Pull-down assays.More importantly,IGF2BP3,as an m6A "Reader",was shown to regulate NRF2 mRNA stability by recognizing m6A modifications of NRF2 mRNA.Similar results were also obtained in in vivo experiments.In summary,our study reveals the regulatory role of IGF2BP3-NRF2 pathway in sorafenib-induced ferroptosis in HCC cells,providing an important theoretical basis for new anti-cancer strategies and drug development.Part Ⅰ.Study of the effect of IGF2BP3 on sorafenib-induced ferroptosis in hepatocellular carcinomaObjectiveExploring the effect of IGF2BP3 on sorafenib-induced ferroptosis in HCC cellsMethods1.The gene expression of IGF2BP3 in HCC and its correlation with HCC prognosis were further explored by mining public gene expression data.2.Construction of Huh7 cell line and Hep3B cell line with knockdown IGF2BP3 using transient transfection method.3.Reactive Oxygen Species(ROS)content was detected using Reactive Oxygen Detection Kit(DCFH-DA fluorescent probe method),and the effect of sorafenib on intracellular ROS content in HCC cells after knockdown of IGF2BP3 was observed using confocal microscopy.4.The effect of sorafenib on intracellular lipid hydroperoxides content in HCC cells after knockdown of IGF2BP3 was observed by using C11 BODIPY581/591(a fluorescent probe for detection of lipid hydroperoxides)staining combined with flow cytometry for quantitative analysis in the presence or absence of Fer-1/Z-VAD-FMK.5.The cell viability assay kit(CCK-8)was applied to detect the growth inhibition of HCC cells and to observe the effect of sorafenib on growth inhibition of HCC cells after knockdown of IGF2BP3,as well as to identify the types of growth inhibition affecting sorafenib-induced HCC cells by using different cell death inhibitors(ferropto sis inhibitor:Fer1;apoptosis inhibitor:Z-VAD-FMK).6.The effect of sorafenib on malondialdehyde(MDA)content in HCC cells after knockdown of IGF2BP3 was observed by using malondialdehyde(MDA)assay kit for the detection of malondialdehyde(MDA),a lipid oxidation end product.7.The effect of sorafenib on iron content in HCC cells after knockdown of IGF2BP3 was observed by using iron assay kit.8.Western Blot was applied to detect the effect of knockdown of IGF2BP3 on the expression of SLC7A11 and GPX4,the key regulatory proteins of ferroptosis,to investigate the effect of knockdown of IGF2BP3 on sorafenib-induced ferroptosis.9.IGF2BP3-knockdown(IGF2BP3-KD)and negative control(NC)Huh7 cell lines were injected subcutaneously for tumorigenic assays in nude mice,respectively.Sorafenib was administered intraperitoneally to nude mice at a dosing rate of 10 mg/kg once every 2 days for 6 weeks.Tumor volume was measured weekly,and finally the weight of collected tumors was measured to observe the effect of knockdown of IGF2BP3 on tumorigenesis in nude mice.10.The proteins of tumor tissues from nude mice were extracted,and the expression of SLC7A11 and GPX4 was examined by using Western Blot assay to observe the effect of knocking down IGF2BP3 on ferroptosis.Results1.IGF2BP3 expression was significantly upregulated in human HCC tumors and portal vein tumor thrombus,and higher levels of IGF2BP3 gene expression were associated with shorter disease-free and overall survival,suggesting that IGF2BP3 may be associated with HCC invasion,early recurrence and poor prognosis.2.The effect of sorafenib on ROS in IGF2BP3-knockdown HCC cells was observed by using DCFH-DA fluorescent probe combined with confocal microscopy.The results showed that the fluorescence intensity of the experimental group with IGF2BP3-knockdown was significantly enhanced compared with the negative control group after sorafenib treatment in both Huh7 and Hep3B cell lines,suggesting a significant increase in ROS content.3.C11 BODIPY 581/591 fluorescent probe combined with flow cytometry was used to quantify the effect of sorafenib on the content of lipid hydroperoxides in IGF2BP3-knockdown HCC cells.The results showed that the intracellular lipid hydroperoxides content was significantly increased in the knockdown IGF2BP3-knockdown group compared with the negative control group after sorafenib treatment,and Fer-1 significantly inhibited this increase in lipid hydroperoxides,while Z-VAD-FMK did not.4.The results of CCK-8 assay showed that knockdown of IGF2BP3 significantly enhanced cell growth inhibition compared with the negative control group in both sorafenib-treated HCC cell lines,and these enhancements of growth inhibition were mostly antagonized by Fer-1,while the antagonistic effect of Z-VAD-FMK was not obvious.5.The results of MDA(an important end product of lipid peroxidation)assay showed that the MDA content in both IGF2BP3-knockdown HCC cells and negative control HCC cells increased after sorafenib treatment,and the MDA content in IGF2BP3-knockdown HCC cells increased more significantly after sorafenib treatment.6.The results of iron content assay showed that the iron content significantly increased in IGF2BP3-knockdown HCC cells after sorafenib treatment,while did not increase significantly in IGF2BP3-knockdown HCC cells without sorafenib treatment.7.Western Blot assay showed that the expression of GPX4 and SLC7A11 decreased in both IGF2BP3-knockdown HCC cells and negative control HCC cells after sorafenib treatment and the expression of GPX4 and SLC7A11 in IGF2BP3-knockdown HCC cells decreased more significantly after sorafenib treatment.8.The weight and volume of tumors in nude mice were measured,and the results showed that the weight and volume of tumors in IGF2BP3-knockdown group were significantly lower than those in the negative control group.Then the results of Western Blot assay on the tumors showed that the expression levels of GPX4 and SLC7A11 in the tumor tissues of the IGF2BP3knockdown group were significantly lower compared with the negative control group.Conclusion1.IGF2BP3 is highly expressed in HCC tumors and portal vein tumor thrombus,and the higher the level of IGF2BP3 gene expression,the more aggressive HCC is,the more likely it is to recur early,and the worse the prognosis.2.Sorafenib can activate ferroptosis in HCC cells.3.Knockdown of IGF2BP3 can significantly promote sorafenib-induced ferroptosis in HCC cells.4.Knockdown of IGF2BP3 can enhance the antitumor efficacy of sorafenib in nude mice by promoting sorafenib-induced ferroptosis in HCC cellsPart Ⅱ Study on the molecular mechanism of IGF2BP3 regulating ferroptosis in hepatocellular carcinomaObjectiveTo explore the downstream regulator and regulatory mechanisms of IGF2BP3 in the regulation of ferroptosisMethods1.Using bioinformatics methods,we used PubMed,KEGG,RBPmap and RBPDB databases to initially predict the mRNAs that potentially bind to IGF2BP3 and participate in the ferroptosis pathway.2.RBP-RIP assay was applied in Huh7 cells to further screen the mRNAs with the highest binding levels from the above prediction results,and then the target mRNAs were further validated by RNA pull-down assay.3.We immunoprecipitated the methylated target mRNA(verified in the first two steps)using anti-m6A antibody in Huh7 and Hep3B cells by MeRIP(methylated RNA immunoprecipitation)assay and assessed the abundance of m6A of this target mRNA by qPCR in HCC cell lines.The effect of knockdown of IGF2BP3 on the expression level of the target mRNA within HCC cells was also assessed by qPCR.4.After applying actinomycin D(Act D)to treat Huh7 and Hep3B cells,we detected the effect of knocking down IGF2BP3 on the expression level of target mRNA at different time points by qPCR.5.We applied bioinformatics methods to first preliminarily predict the potential m6A binding sites of IGF2BP3 to target mRNAs using the website tools RBPmap and SRAMP website tools.The predicted potential m6A sites of the target mRNA were base mutated and verified by dual luciferase assay to determine the specific m6A binding sites of IGF2BP3 to the target mRNA.6.Rescue assay:Western Blot assay was applied to detect the changes in the expression levels of target protein,ferroptosis key regulatory protein SLC7A11 and GPX4 after knockdown of IGF2BP3 and overexpression of target protein after knockdown of IGF2BP3 in Huh7 and Hep3B cell lines to clarify whether IGF2BP3 affects ferroptosis levels by affecting this downstream target protein to affect the level of ferroptosis.7.The proteins from tumor tissues of nude mice were extracted and the expression of target protein,SLC7A11 and GPX4 were examined by applying Western Blot to observe the effect of knocking down IGF2BP3 on target protein as well as ferroptosis.Results1.The top four transcripts in terms of binding capacity were initially predicted and screened by cross-analysis of PubMed,KEGG,RBPmap and RBPDB databases.2.Application of RNA immunoprecipitation(RBP-RIP)assay revealed the highest content of NRF2 mRNA in anti-IGF2BP3-mRNA complex,suggesting that IGF2BP3 binds most readily to NRF2 mRNA and IGF2BP3 may be a regulatory protein of NRF2.3.In the RNA pull-down assay,the NRF2 mRNA probe was significantly enriched for IGF2BP3 protein,indicating the binding of IGF2BP3 and NRF2 mRNA,suggesting that IGF2BP3 may be involved in the regulation of NRF2.4.MeRIP(methylated RNA immunoprecipitation)experiments followed by qPCR evaluation showed that NRF2 mRNA was abundantly modified by m6A in both Huh7 and Hep3B cell lines.qPCR results also showed that knockdown of IGF2BP3 significantly reduced the expression level of NRF2 mRNA.5.The synthesis of new RNA in cells with NC or knockdown IGF2BP3 was blocked with Act D(actinomycin D,1 μg/ml)and the decay of mRNA was assessed at different time points.The levels of NRF2 mRNA were found to be significantly decreased in both knockdown IGF2BP3 HCC cells compared to the control group.These suggest that IGF2BP3 may influence the expression level of NRF2 mRNA by affecting its stability.6.We applied a bioinformatics approach to predict two m6A binding sites of IGF2BP3 to NRF2 mRNA by RBPmap and SRAMP database tools.The binding sites were located at+1482(from the start codon)or+1486 bases in the CDS(coding sequence)region.The+1482 base of NRF2 mRNA was verified to be the most likely binding site for IGF2BP3 by a dual luciferase assay.7.Western blot assay confirmed that overexpression of NRF2 in HCC cells could significantly restore the reduced levels of GPX4 and SLC7A11(markers of ferroptosis)caused by IGF2BP3 knockdown.8.The results of Western Blot assay showed that the expression levels of NRF2,GPX4 and SLC7A11 were significantly reduced in the tumor tissues of nude mice in the knockdown IGF2BP3 experimental group compared with the negative control group.Conclusion1.IGF2BP3 can bind to the NRF2 mRNA.2.IGF2BP3 may regulate NRF2 expression by affecting the stability of NRF2 mRNA.3.IGF2BP3 may regulate the stability of NRF2 mRNA by binding to the m6A modification site of NRF2 mRNA.4.IGF2BP3 may affect ferroptosis in HCC by regulating the expression of NRF2. |
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