RNA Binding Protein DAZAP1 Promotes HCC Progression And Regulates Ferroptosis By Interacting With SLC7A11 MRNA

Objective:Hepatocellular carcinoma(HCC)is the sixth most commonly diagnosed cancer and the third leading cause of cancer death worldwide.The numbers of newly diagnosed cases and deaths in 2012 were predicted to be 782,500 and 745,500,which rose to 906,000 and 830,000,respectively,in 2020,It is broadly believed that HCC has an insidious onset,low early diagnosis rate,rapid progression and high malignancy,causing most patients were already in advanced stages at the time of diagnosis and lost the opportunity for surgery.Therefore,clarifying the mechanism of the occurrence and development of HCC and exploring and researching new molecular targets are essential for improving its early diagnosis rate,prolonging patient survival and improving patient prognosis.RNA binding proteins(RBPs),a class of proteins that bind to coding or noncoding transcripts,participate in nearly the entire lifecycle from production to degradation of RNAs.For these reasons,it is important to fully understand the regulatory networks of RBPs and their binding partners.DAZAP1 is a widely and abundantly expressed RBP.It was initially identified as an important binding partner of DAZ(deleted in azoospermia)and DAZL(deleted in azoospermia like)involved in not only spermatogenesis but also the normal growth and development of mammals.It is rarely studied in tumors,and only reported in few malignant tumors such as lung cancer and leukemia.This study aims to reveal the role of DAZAP1 in the occurrence and development of HCC and sorafenib-mediated ferroptosis,and to provide new molecular targets and theoretical foundations for the early diagnosis,improvement of treatment and prognosis of HCC.Methods:1.Exploring the expression of DAZAP1 in HCC cells and tissues qRT-PCR and Western Blot was used to detect the expression level of DAZAP1 in normal liver cells(L02)and five HCC cell lines(HepG2,SMMC-7721,Hep3B,Bel-7402,Huh7).And then,we detected the protein expression level of DAZAP1 in HCC tissues and adjacent tissues by Western Blot and IHC.Meanwhile,we screened the survival curve of patients with HCC related to the expression level of DAZAP1 in TCGA(The Cancer Genome Atlas)database.2.Exploring the effect of DAZAP1 on the proliferation,invasion and migration of HCC cellsSmall interfering RNA against DAZAP1(si-DAZAP1)and overexpression plasmids of DAZAP1(oe-DAZAP1)were used to transfect HCC cell lines(HepG2 and Hep3B)respectively,and the transfection efficiency was detected by qRT-PCR.We use CCK-8(Cell Counting Kit 8),EdU(5-Ethynyl-2’-deoxyuridine)and Cell clone formation assays to detect the effects of knocking down or overexpressing DAZAP1 on the proliferation of HepG2 and Hep3B cells.The Transwell assay was used to explore the effects of DAZAP1 knockdown or overexpression on the invasion or migration of HCC cells.3.Exploring the effect of DAZAP1 on the ferroptosis of HCC cellsqRT-PCR and Western Blot were used to detect the expression of DAZAP1 under the influence of sorafenib,the first-line targeted drug for HCC,in HepG2 and Hep3B cells.Different cell death inhibitors(Ferroptosis inhibitor:Ferrostatin-1;Apoptosis inhibitor:Z-VAD-FMK;Cell death inhibitor:Necrosulfonamide)were used to identify the types of cell death,among which the known ferroptosis agonist,Erastin,was compared with sorafenib.The level of ferroptosis in HCC cells was determined by detecting intracellular iron,glutathione(GSH),and malondialdehyde(MDA).4.Explore and verify ferroptosis pathway targets that can interact with DAZAP 1Through bioinformatics screening and comparison,it is found that the mRNA of the key protein SLC7A11(Solute Carrier Family 7 Member 11)in the ferroptosis pathway is a potential target of DAZAP1.qRT-PCR and Western Blot were used to detect the impact of DAZAP1 knockdown on the ferroptosis pathway genes(SLC7A11,SLC3A2(Solute Carrier Family 3 Member 2),GCL(Glutamate-Cysteine Ligase Catalytic Subunit),GSS(Glutathione Synthetase),ACSL4(Acyl-CoA Synthetase Long Chain Family Member 4),LPCAT3(Lysophosphatidylcholine),ALOX15(Arachidonate 15-Lipoxygenase)and GPX4(Glutathione peroxidase 4)).The relationship between DAZAP1 and SLC7A11 mRNA was determined by Dual-luciferase gene reporter and RNA binding protein immunoprecipitation(RIP)assays.5.Exploring the effect of DAZAP1 on the SLC7A11-mediated ferroptosis glutathione metabolism pathwayWe explored deeply the impact of DAZAP1 knockdown on the expression of SLC7A11 mRNA by Act D(Actinomycin D)and Agarose gel electrophoresis assays.qRT-PCR and Western Blot assays were used to explore the effect of DAZAP1 on SLC7A11 and GPX4 expression levels in the ferroptosis pathway.6.Rescuing experiments to verify the role of DAZAP1 in ferroptosissi-SLC7A11 was used to transfect HepG2 and Hep3B cells,and the transfection efficiency was detected by qRT-PCR and Western Blot assays.The CCK-8 assay was used to detect the impact of co-transfection of oe-DAZAP1 and si-SLC7A11 on the growth inhibition of HCC cells.The effects of co-transfection of oe-DAZAP1 and si-SLC7A11 on ferroptosis were determined by detecting the ferroptosis related indicators(Iron,GSH and MDA)of HCC.7.Verifying the effect of DAZAP1 on ferroptosis in vivoThe subcutaneous tumor formation experiment in male nude mice was used to verify the effect of DAZAP1 on the size of tumors.qRT-PCR and Western Blot assays were used to further explore the effect of DAZAP1 on the expression of SLC7A11 and GPX4 in ferroptosis pathway in vivo.Results:1.For the expression level of DAZAP1 in cells,the results of qRT-PCR and Western Blot showed that the normal liver cell(L02)was much lower than five HCC cell lines(HepG2,SMMC-7721,Hep3B,Bel-7402,Huh7).For the expression of DAZAP1 in tissues,the results of Western Blot and immunohistochemical(IHC)staining assays revealed that the HCC tissues were higher than corresponding adjacent tissues.And then,TCGA database survival curve indicated that patients with high expression of DAZAP1 have a poorer prognosis than those with low expression.2.The results of DAZAP1 functional experiments of HCC cells showed that DAZAP1 played a positive regulatory role on HCC cells.Compared with the negative control(NC)group,after knocking down DAZAP1,the proliferation,invasion and migration ability of HepG2 and Hep3B cells were significantly reduced.When DAZAP1 was overexpressed,the results were opposite.3.Sorafenib played a positive regulator role on DAZAP1 in HCC cells.It was verified by qRT-PCR and Western Blot assays.The results of CCK-8 assays showed that under the action of sorafenib,knocking down DAZAP1 significantly increased the level of growth inhibition of HCC cells.Meanwhile,compared with apoptosis inhibitor(Z-VAD-FMK)and cell death inhibitor(Necrosulfonamide),ferroptosis inhibitor(Ferrostatin-1)significantly down-regulated the growth inhibition of HCC cells under the application of Erastin or sorafenib.For the ferroptosis-related indicators,with the knockdown of DAZAP1,cellular iron and MDA were accumulated and endogenous GSH was decreased under the influence of Erastin or Sorafenib.4.Bioinformatics analysis and screening revealed that SLC7A11 mRNA is a potential target of DAZAP1.qRT-PCR and Western Blot results showed that the expression of SLC7A11 was down-regulated as DAZAP1 knockdown.RIP results revealed that DAZAP1 can bind to SLC7A11 mRNA.The luciferase gene report showed that overexpression of DAZAP1 can significantly increase luciferase activity in HCC cells transfected with wide type(WT)SLC7A11 mRNA sequence,while mutant SLC7A11 mRNA sequence had not this effect.5.ActD and Agarose gel electrophoresis experiments showed that DAZAP 1 knockdown in HCC cells can significantly reduce the stability of SLC7A11 mRNA.There were two controls(DMSO group with sorafenib group,sorafenib+NC group with sorafenib+si-DAZAP1 group)to exploring the expression of DAZAP1,SLC7A11 and GPX4 in ferroptosis pathway.(1)The qRT-PCR results showed that compared with the DMSO group,when HCC cells were only administered with sorafenib,DAZAP1 and SLC7A11 were up-regulated,while the expression of GPX4 was down-regulated.And compared with sorafenib+NC group,the expressions of DAZAP1,SLC7A11 and GPX4 were significantly decreased when HCC cells were simultaneously administered with sorafenib and DAZAP1 knockdown.(2)Western Blot results showed that compared with the DMSO group,when only sorafenib was administered to HCC cells,the expression of DAZAP1 was up-regulated,and SLC7A11 and GPX4 expressions were down-regulated.And the results of sorafenib+si-DAZAP1 group were similar to qRT-PCR.6.Rescuing experiments showed that si-SLC7A11 could restore the reduction in cell growth inhibition caused by overexpression of DAZAP1,and it can also restore the ferroptosis-related indicators of HCC cells(cellular Iron,GSH and MDA).7.The results of experiments in vivo showed that knocking down DAZAP1 significantly reduced the volume and mass of subcutaneous tumors in nude mice.At the same time,the experiments verified that DAZAP1 affects the ferroptosis glutathione metabolism pathway,the results were the same as experiments in vitro.Conclusion:1.As a ubiquitous RBP,DAZAP1 is highly expressed in HCC cells and tissues,and the expression level of DAZAP 1 is related to the prognosis of HCC patients.2.DAZAP1 could promote the proliferation,invasion and migration of HCC.3.DAZAP1 could antagonize the activation of ferroptosis by sorafenib.4.In terms of mechanism,DAZAP1 binds to the SLC7A11 mRNA in ferroptosis glutathione metabolism pathway,thereby regulating the function of GPX4 and inhibiting ferroptosis of HCC.Significance:This study focused on a RBP,DAZAP1,and analyzed its expression in HCC cells and tissues and the relationship between its expression level with the prognosis of HCC patients.Then we further explored the effect of DAZAP1 on the proliferation,invasion,migration and sorafenib-mediated ferroptosis in HCC.And we also analyze the specific molecular mechanism deeply of DAZAP1 affecting ferroptosis pathway.Therefore,this study is of great significance for the diagnosis and treatment of HCC,improving the efficacy of sorafenib and improving the prognosis of patients.