HO-1 Complexes With 14-3-3ζ To Promote Hepatocellular Carcinoma Progression

Objective: To investigate the role of HO-1 in HCC progression,evaluate the association between the expression of HO-1 and the clinicopathologic features of HCC tissues and analyze the prognostic of HO-1 in HCC,two independent cohorts of HCC patients were used in this study.Material and Methods:(1)For the first cohort(cohort 1,snap-frozen tissues),70 paired HCC samples,obtained from patients undergoing curative resection at Tongji Hospital of Huazhong University of Science and Technology(Wuhan,China),were used for quantitative real-time PCR(q RT-PCR)and western blot analysis.We divided the samples into groups,based on the stage of tumor development,and then studied the differences in HO-1 expression among these groups.(2)For cohort 2,a tissue microarray-based IHC study of HO-1 was performed on 148 HCC tissues from a cohort with comparable clinicopathological features and available follow-up data.Prognostic value of HO-1 and other clinicopathologic factors were evaluated.Results:(1)We found that HO-1 m RNA and protein levels in patients with advanced Barcelona Clinic Liver Cancer(BCLC)-C stage HCC were considerably higher than those patients with Barcelona Clinic Liver Cancer(BCLC)-A or-B stage HCCs in cohort 1.(2)By examining the correlation between HO-1 expression in HCC tissues and clinicopathological features,we found that HO-1 expression was lower in low-BCLC stage tumors,and higher in high-BCLC stage tumors in cohort 2.In addition,the data showed that HO-1 expression was closely associated with several aggressive clinicopathologic features,including incomplete tumor encapsulation,positive microvascular invasion,advanced TNM stage,and advanced BCLC stage.A Kaplan-Meier’s analysis showed that patients with high HO-1 expression had poorer overall survival(log rank,7.356;P = 0.006)and disease-free survival rates(log rank,7.016;P = 0.008)than patients with low HO-1 expression.Conclusion: Increased HO-1 expression correlates with aggressive clinicopathologic features and predicts poor prognosis in HCC patients,suggesting HO-1 may play an important role in HCC progression.Objectives: To explore the function roles of HO-1 in hepatocellular carcinoma progression using both in vitro and in vivo assays.Material and Methods:(1)we first investigated the endogenous HO-1 levels in ten HCC cell lines with different metastatic potentials.We then constructed HO-1-KO Bel7402 cells using the CRISPR-Cas9 system.We also generated a stable HO-1 knockdown Bel7402 cell line,and a stable HO-1 overexpressing HLF cell line.(CCK-8)proliferation assay,colony formation assay,in vivo tumorigenicity assays were performed to examined the effect of HO-1 on the cell growth and tumorigenicity of HCC cells.A cell-cycle analysis was used to assess whether HO-1 could affect cell cycle progression.(2)In vitro migration / invasion assays,in vivo metastasis assays,bioluminescent imaging,HE staining were performed to investigate the role of HO-1 in tumor metastasis.Result:(1)We found significantly increased HO-1 levels in Bel7402,MHCC-97 L,MHCC-97 H,and HCC-LM3 cell lines,which have higher invasive and metastatic capabilities,compared with cell lines with lower metastatic potentials.Knockout of HO-1 significantly inhibited cell proliferation.Similarly,a knockdown of HO-1 in Bel7402 cells decreased cell proliferation.In contrast,overexpression of HO-1 promoted cell growth.In a subcutaneous implantation nude mice model,nude mice injected with HO-1 knockout or knockdown Bel7402 cells showed significantly decreased tumor growth.In contrast,overexpression of HO-1 promoted the in vivo tumor growth of HLF cells.The number of G1-phase cells was significantly increased,whereas the number of S-phase cells was significantly decreased,when HO-1 was knocked-out or knocked-down.In contrast,overexpression of endogenous HO-1 increased G1-to S-phase cell-cycle progression.(2)the migratory and invasive abilities of Bel7402 cells were significantly impaired when HO-1 expression was either knocked-out or knocked-down.In contrast,upregulation of HO-1 increased the migration and invasiveness of HLF cells.HO-1 knockout or knockdown significantly inhibit in vivo lung metastases,while overexpression of HO-1 in HLF cells significantly promoted in vivo lung metastases.Conclusion:(1)HO-1 promotes HCC growth.(2)HO-1 expression not only induces migration and invasion of tumor cells in vitro,but also promotes their ability to metastasize in vivo.Objective: To elucidate the molecular mechanisms by which HO-1 promotes HCC proliferation and metastasis,we attempted to screen for its interacting partners.Our current study attempted to explore the possible involvement of upstream or downstream effectors in HO-1-mediated tumor progression in HCC.We demonstrated that HO-1 complexes with 14-3-3ζ to promote hepatocellular carcinoma progression.As follows:(1)HO-1 interacts with 14-3-3ζ(2)14-3-3ζ stabilizes the HO-1 protein by inhibiting its ubiquitination(3)HO-1 is implicated in 14-3-3ζ-mediated tumor proliferation and migration/invasion.Methods:(1)HEK293 cells were transfected with a Flag-tagged HO-1 construct and the anti-Flag immunoprecipitates were separated by SDS-PAGE,silver-stained,and protein bands were analyzed by mass spectrometry to creen for its interacting partners.Co-immunoprecipitation(co-IP)was performed to demonstrate the interactions of exogenous HO-1 with 14-3-3ζ and determine whether other 14-3-3 isoforms could also interact with HO-1.Immunofluorescence confocal microscopy was performed to reveal the colocalization of endogenously expressed HO-1 and 14-3-3ζ.A series of Flag-tagged HO-1 deletion mutants were created to identify which region of HO-1 responsible for the 14-3-3ζ interaction.(2)Western blot,and RT-PCR assays were performed to demonstrate that knockdown of 14-3-3ζ significantly reduced the protein expression of HO-1,but had no effect on m RNA levels.Degradation assay,in vivo ubiquitination assay were performed to demonstrate that 14-3-3ζ stabilizes the HO-1 protein by inhibiting its ubiquitination.(3)In vitro proliferation assay,a colony formation assay,migration/invasion assay were performed to demonstrate that 14-3-3ζ knockdown in HLF cells stably overexpressing HO-1 reversed HO-1 enhanced cell proliferation,migration/invasion.We performed small interfering RNA(si RNA)-mediated HO-1,Western blot,in vitro proliferation assay,colony formation assay,migration/invasion assay demonstrate that 14-3-3ζ overexpression-induced increases in cell proliferation and migration/invasion capabilities were abolished by transfection with a HO-1-specific si RNA.Result:(1)A analysis of the MS data indicated that the 14-3-3 proteins were potential HO-1 interacting partners.Co-IP assay showed that 14-3-3ζ interacts with HO-1 to a greater extent than the other isoforms.Immunofluorescence confocal microscopy revealed the colocalization of endogenously expressed HO-1 and 14-3-3ζ in the cytoplasm.Co-IP assay showed that the region encompassing amino acids 265-288 in HO-1 was essential for the interaction with 14-3-3ζ.(2)Degradation assay showed that knockdown of 14-3-3ζ expression significantly reduced the half-life of HO-1.The downregulation of HO-1 in 14-3-3ζ-knockdown cells was restored in the presence of the proteasome inhibitor MG132,indicating that 14-3-3ζ-knockdown facilitates the degradation of HO-1.Ubiquitination of HO-1 was found to be increased in Bel7402 and HLF cells with a 14-3-3ζ knockdown,compared to control cells.(3)14-3-3ζ knockdown in HLF cells stably overexpressing HO-1 reversed HO-1 enhanced cell proliferation,migratory and invasive abilities.The 14-3-3ζ overexpression-induced increases in cell proliferation and migration/invasion capabilities were abolished by transfection with a HO-1-specific si RNA.Conclusion:(1)these data demonstrate that HO-1 could associate with 14-3-3ζ,and that the interaction was mediated through a disordered region of HO-1 rather than through phosphorylation of a motif.(2)These data indicate that 14-3-3ζ maintains HO-1 levels primarily through inhibiting its ubiquitination.(3)HO-1 is implicated in 14-3-3ζ-mediated tumor proliferation and migration/invasion.